Method for the production of calendic acid, a fatty acid containing delta-8,10,12 conjugated double bonds and related fatty acids having a modification at the delta-9 position

ABSTRACT

The preparation and use of nucleic acid fragments encoding plant fatty acid modifying enzymes associated with modification of the delta-9 position of fatty acids, in particular, formation of conjugated double bonds are disclosed. Chimeric genes incorporating such nucleic acid fragments and suitable regulatory sequences can be used to create transgenic plants having altered lipid profiles. The preparation and use of nucleic acid fragments encoding plant fatty acid modifying enzymes associated with formation of a trans delta-12 double bond also are disclosed. Chimeric genes incorporating such nucleic acid fragments and suitable regulatory sequences can be used to create transgenic plants having altered lipid profiles.

This application claims priority benefit of U.S. Provisional ApplicationNo. 60/149,050 filed Aug. 16, 1999, now abandoned.

FIELD OF THE INVENTION

This invention relates to fatty acid biosynthesis and, in particular, tothe preparation and use of nucleic acid fragments encoding plant fattyacid modifying enzymes associated with modification of the delta-9position of fatty acids and, in particular, formation of conjugateddouble bonds. Chimeric genes incorporating such nucleic acid fragmentsand suitable regulatory sequences can be used to create transgenicplants having altered lipid profiles. This invention also relates to thepreparation and use of nucleic acid fragments encoding plant fatty acidmodifying enzymes associated with the formation of a trans-delta-12double bond. Chimeric genes incorporating such nucleic acid fragmentsand suitable regulatory sequences can be used to create transgenicplants having altered lipid profiles.

BACKGROUND OF THE INVENTION

Fatty acids bearing chemical modifications in addition to the commondouble bonds are found in the storage lipids of many oilseeds (Badamiand Patil (1981) Prog. Lipid Res. 19:119-153). Some of thesemodifications functionalize the fatty acid to produce products that areuseful in industrial applications; this is an alternative to the morecommon usage of plant-derived lipids as foods. Examples are the use ofthe hydroxylated fatty acid ricinoleic acid in lubricants, and theshort- or medium-carbon chain length fatty acids from palm oil indetergents. In some cases, fatty acid composition of the storage lipidsof oilseeds produced in temperate climates can be modified by theaddition of genes from exotic sources so that large amounts of uniquefatty acids are produced (Ohlrogge, J. B. (1994) Plant Physiol. 104,821-826).

Fatty acids containing conjugated double bonds are major components ofthe seed oil of a limited number of plant species. For example, calendicacid (8-trans, 10-trans, cis-12-octadecatrienoic acid) composes greaterthan 50% of the total fatty acids of the seed oil of Calendulaofficinalis (Crombie and Holloway (1984) J. Chem. Soc. Chem. Commun. 15,953-955, Chisholm, M. J. & Hopkins, C. Y. (1967) Can. J. Biochem45:251-254). Another example, α-parinaric acid (9-cis, 11-trans,13-trans, 15-cis-octadecatetraenoic acid) and β-parinaric acid (9-trans,11-trans, 13-trans, 15-cis-octadecatetraenoic acid) compose more than25% of the total fatty acids of the seed oil of Impatiens species(Bagby, M. O., Smith, C. R. and Wolff, I. A. (1966) Lipids 1, 263-267).In addition, α-eleostearic acid (9-cis, 11-trans,13-trans-octadecatrienoic acid) and β-eleostearic acid (9-trans,11-trans, 13-trans-octadecatrienoic acid) compose >55% of the totalfatty acids of the seed oil of Momordica charantia (Chisolm, M. J. andHopkins, C. Y. (1964) Can. J. Biochem. 42, 560-564; Liu, L., Hammond, E.G. and Nikolau, B. J. (1997) Plant Physiol. 113, 1343-1349). Calendicacid and eleostearic acid are both 18:3 fatty acids, like linolenicacid, however, their structures are quite different, as shown in FIG. 1.Another fatty acid containing conjugated double bonds is found in theseeds of Dimorphotheca sinuata. This unusual C₁₈ fatty acid,dimorphecolic acid (9-OH-18:2Δ^(10trans,12trans)), contains twoconjugated trans-double bonds between the Δ¹⁰ and Δ¹¹ carbon atoms andbetween the Δ¹² and Δ¹³ carbon atoms as well as a hydroxyl group on theΔ⁹ carbon atom [Binder, R. G. et al., (1964) J. Am. Oil Chem. Soc.41:108-111; Morris, L. J. et al., (1960)J. Am. Oil Chem. Soc.37:323-327]. Thus, there are certain 18:2 and 18:3 plant fatty acidsthat contain conjugated double bonds.

The presence of conjugated double bonds in fatty acids provides thefunctional basis for drying oils such as tung oil that are enriched inisomers of eleostearic acid. This is due largely to the fact that fattyacids with conjugated double bonds display high rates of oxidation,particularly when compared to polyunsaturated fatty acids with methyleneinterrupted double bonds. Drying oils, such as tung oil, are used ascomponents of paints, varnishes, and inks.

Conjugated fatty acids can also be used as an animal feed additive.Conjugated linoleic acids (CLAs, 18:2) have been used to improve fatcomposition in feed animals.

U.S. Pat. No. 5,581,572, issued to Cook et al. on Dec. 22, 1998,describes a method of increasing fat firmness and improving meat qualityin animals using conjugated linoleic acds.

U.S. Pat. No. 5,554,646, issued to Cook et al. on Sep. 10,1996,describes a method of reducing body fat in animals using conjugatedlinoleic acids.

U.S. Pat. No. 5,519,451, issued to Cook et al. on Jul. 6, 1999,describes a method of improving the growth or the efficiency of feedconversion of an animal which involves animal feed particles having aninner core of nutrients and an outer layer containing a conjugated fattyacid or an antibody that can protect the animal from contacting diseasesthat can adversely affect the animal's ability to grow or efficientlyconvert its feed into body tissue.

U.S. Pat. No. 5,428,072, issued to Cook et al. on Jun. 27, 1995,describes a method of enhancing weight gain and feed efficiency inanimals, which involves the use of conjugated linoleic acid.

The mechanism by which these effects are realized is not known. It isbelieved that no one heretofore has discussed the use of conjugated 18:3fatty acids (conjugated linolenic acids or ClnAs), for improving animalcarcass characteristics.

The biosynthesis of fatty acids with conjugated double bonds is not wellunderstood. Several reports have indicated that conjugated double bondsare formed by modification of an existing double bond (Crombie, L. andHolloway, S. J. (1985) J. Chem. Soc. Perkins Trans. I 1985, 2425-2434;Liu, L., Hammond, E. G. and Nikolau, B. J. (1997) Plant Physiol. 113,1343-1349). For example, the double bonds at the 11 and 13 carbon atomsin eleostearic acid have been shown to arise from the modification ofthe Δ¹² double bond of linoleic acid (18:2Δ^(9,12)) (Liu, L., Hammond,E. G. and Nikolau, B. J. (1997) Plant Physiol. 113, 1343-1349). Theexact mechanism involved in conjugated double formation in fatty acids,however, has not yet been determined. Fatty acid desaturase(Fad)-related enzymes are responsible for producing 18:3Δ^(9,11,13) oilssuch as α and β-eleostearic acid and 18:4Δ^(9,11,13,15) oils such as αand β-parinaric acid in Impatiens, Momordica, and Chrysobalanus.Insertion of a chimeric gene comprising an isolated nucleic acidfragment encoding these enzymes into species that do not normallyaccumulate conjugated double-bond containing; fatty acids resulted inproduction of eleostearic and/or parinaric acids (Cahoon et al. (1999)Proc. Natl. Acad. Sci. USA 96:12935-12940; and WO 00/11176, published onMar. 2, 2000, the disclosure of which is hereby incorporated byreference). The present invention extends this work by answering whether18:3Δ^(8,10,12) fatty acids like calendic or dimorphecolic acids canalso be produced in transgenic plants. Unlike the Fad-related enzymesthat modify the delta-12 position to produce eleostearic and parinaricacids, the enzymes of the present invention (with one exception as isdiscussed below with respect to DMFad2-1) modify the delta-9 position offatty acids to produce calendic and dimorphecolic acids. One enzyme isdisclosed herein which is associated with the formation of atrans-delta-12 double bond. The product of this enzymatic reaction thenbecomes the substrate for a reaction involving conjugated double bondformation comprising a delta-9 position of fatty acids. Isolation andcharacterization of two Calendula cDNAs, two Dimorphotheca cDNAs, andexpression of a chimeric transgene, are described herein.

SUMMARY OF THE INVENTION

This invention concerns an isolated nucleic acid fragment encoding aplant fatty acid modifying enzyme associated with conjugated double bondformation comprising a delta-9 position of fatty acids wherein saidfragment or a functionally equivalent subfragment thereof (a) hybridizesto any of the nucleotide sequences set forth in SEQ ID NOs:1, 3, or 12under conditions of moderate stringency or (b) is at least 40% identicalto a polypeptide encoded by any of the nucleotide sequences set forth inSEQ ID NOs:1, 3, or 12 or a functionally equivalent subfragment thereofas determined by a comparison method designed to detect homologoussequences.

In a second aspect, this invention concerns an isolated nucleic acidfragment encoding a plant fatty acid modifying enzyme associated withconjugated double bond formation comprising a delta-9 position of fattyacids wherein said fragment, or a functionally equivalent subfragmentthereof, encodes a protein comprising any one of the amino acidsequences set forth in SEQ ID NOs:2, 4, or 13.

In a third aspect, this invention concerns a chimeric gene comprisingsuch isolated nucleic acid fragments, or a functionally equivalentsubfragment thereof, or a complement thereof, operably linked tosuitable regulatory sequences.

In a fourth aspect, this invention concerns a transformed host cell orplant comprising such a chimeric gene.

In a fifth aspect, this invention concerns a method of altering thelevel of fatty acids in a host cell or plant wherein said fatty acidscomprise a modification at a delta-9 position, said method comprising:

(a) transforming a host cell or plant with a chimeric gene as discussedabove;

(b) growing the transformed host cell or plant under conditions suitablefor the expression of the chimeric gene; and

(c) selecting those transformed host cells or plants having alteredlevels of fatty acids with double bonds.

In a sixth aspect, this invention concerns a method for producing seedoil containing fatty acids comprising a modified delta-9 position in theseeds of plants which comprises:

(a) transforming a plant cell with such a chimeric gene;

(b) growing a fertile mature plant from the transformed plant cell ofstep (a);

(c) screening progeny seeds from the fertile plants of step (b) foraltered levels of fatty acids comprising a modified delta-9 position;and

(d) processing the progeny seed of step (c) to obtain seed oilcontaining altered levels of plant fatty acids comprising a modifieddelta-9 position.

In a seventh aspect, this invention concerns a method for producingplant fatty acid modifying enzymes associated with modification of adelta-9 position of fatty acids which comprises:

(a) transforming a microbial host cell with the claimed chimeric genes;

(b) growing the transformed host cell under conditions suitable for theexpression of the chimeric gene; and

(c) selecting those transformed host cells containing altered levels ofprotein encoded by the chimeric gene.

In an eighth aspect, this invention concerns a method to isolate nucleicacid fragments and functionally equivalent subfragments thereof encodinga plant fatty acid modifying enzyme associated with modification of adelta-9 position of fatty acids comprising:

(a) comparing SEQ ID NOs:2, 4, or 13 and other plant fatty acidmodifying enzyme polypeptide sequences;

(b) identifying conserved sequences of 4 or more amino acids obtained instep (a),

(c) designing degenerate oligomers based on the conserved sequencesidentified in step (b); and

(d) using the degenerate oligomers of step(s) to isolate sequencesencoding a plant fatty acid modifying enzyme or a portion thereofassociated with modification of the delta-9 position of fatty acids bysequence dependent protocols.

In an ninth aspect, this invention concerns an isolated nucleic acidfragment encoding a plant fatty acid modifying enzyme wherein saidenzyme modifies a delta-9 position of fatty acids and further whereinsaid fragment or a functionally equivalent subfragment thereof (a)hybridizes to any of the nucleotide sequences set forth in SEQ ID NOs:1,3, or 12 under, conditions of moderate stringency or (b) is at least 40%identical to a polypeptide encoded by any of the nucleotide sequencesset forth in SEQ ID NOs:1, 3, or 12 or a functionally equivalentsubfragment thereof as determined by a comparison method designed todetect homologous sequences.

In an tenth aspect, this invention concerns an isolated nucleic acidfragment encoding a plant fatty acid modifying enzyme wherein saidenzyme modifies a delta-9 position of fatty acids and further whereinsaid fragment or a functionally equivalent subfragment thereof encodes aprotein comprising any one of the amino acid sequences set forth in SEQID NOs:2, 4, or 13.

In a eleventh aspect, this invention concerns isolated nucleic acidfragment encoding a plant fatty acid modifying enzyme wherein saidenzyme modifies a delta-9 position of fatty acids wherein said fragmentor a functionally equivalent subfragment thereof (a) hybridizes to theisolated nucleic acid fragment of claim 2 under conditions of moderatestringency or (b) is at least 40% identical to a polypeptide encoded byany of the isolated nucleic acid fragments of claim 2 or a functionallyequivalent subfragment thereof as determined by a comparison methoddesigned to detect homologous sequences.

Also of interest are chimeric genes comprising such isolated nucleicacid fragments, or a functionally equivalent subfragment thereof, or acomplement thereof, operably linked to suitable regulatory sequences.Transformed host cells or plants comprising such chimeric genes are ofinterest. Indeed, these nucleic acid fragments can be used in any of theabove-identified methods such as altering the level of fatty acids in ahost cell or plant, producing plant fatty acid modifying enzymesassociated with modification of a delta-9 position of a fatty acid, etc.

In a twelfth aspect, this invention concerns an animal feed comprisingan ingredient derived from the processing of any of the seeds obtainedfrom plants transformed with the chimeric genes discussed herein and amethod of improving the carcass quality of an animal by supplementingthe diet of the animal with such animal feeds.

In a thirteenth aspect, this invention concerns an isolated nucleic acidfragment encoding a plant fatty acid modifying enzyme associated withthe formation of a trans delta-12 double bond wherein said enzymemodifies a delta-12 position of fatty acids and further wherein saidfragment or a functionally equivalent subfragment thereof (a) hybridizesto any of the nucleotide sequences set forth in SEQ ID NO:10 underconditions of moderate stringency or (b) is at least 75% identical to apolypeptide encoded by any of the nucleotide sequences set forth in SEQID NO:10 or a functionally equivalent subfragment thereof as determinedby a comparison method designed to detect homologous sequences.

Also of interest are chimeric genes comprising such isolated nucleicacid fragments, or a functionally equivalent subfragment thereof, or acomplement thereof, operably linked to suitable regulatory sequences.Transformed host cells or plants comprising such chimeric genes are ofinterest. Indeed, these nucleic acid fragments can be used in any of theabove-identified methods such as altering the level of fatty acids in ahost cell or plant, producing plant fatty acid modifying enzymesassociated with modification of a delta-12 position of a fatty acid,etc.

BRIEF DESCRIPTION OF THE FIGURES AND SEQUENCE DESCRIPTIONS

The invention can be more fully understood from the following detaileddescription and the Figure and Sequence Descriptions which form a partof this application.

The sequence descriptions summarize the Sequences Listing attachedhereto. The Sequence Listing contains one letter codes for nucleotidesequence characters and the three letter codes for amino acids asdefined in the IUPAC-IUB standards described in Nucleic Acids Research13:3021-3030 (1985) and in the Biochemical Journal 219 (No. 2):345-373(1984), and the symbols and format used for all nucleotide and aminoacid sequence data further comply with the rules governing nucleotideand/or amino acid sequence disclosures in patent applications as setforth in 37 C.F.R. §1.821-1.825 and WIPO Standard St.25.

FIG. 1 shows the structures of α-linolenic acid, calendic acid, andα-eleostearic acid.

FIGS. 2A-2D show a comparison of the amino acid sequences of the instantfatty acid modifying enzymes associated with conjugated double bondformation comprising a modification of the delta-9 position of fattyacids from seeds of Calendula officinalis (CalFad2-1 and CalFad2-2),Dimorphotheca sinuata (DMFad2-2), and a delta-12 modifying enzyme fromDimorphotheca sinuata (DMFad2-1). The two Calendula genes, that encodeenzymes that form 18:3□^(8,10,12) conjugated double bonds, are comparedto the genes from Impatiens balsamina (ImpFad2 H8), Momordica charantia(MomFad2), and Chrysobalanus icaco (ChrFad2) that encode enzymes forming18:3□^(9,11,13) conjugated double bonds, a castor bean fatty acidhydroxylase (Hydroxylase), and a soybean omega-6 oleate desaturase (Soyomega-6). The two Dimorphotheca sinuata amino acid sequences (DMFad2-1and DMFad2-2) are compared to delta-12 fatty acid desaturases fromsunflower (Helianthus annuus), and borage (Borago offlicinalis),respectively. The conserved histidine motifs found in desaturases andhydroxylases are boxed. The position of the glycine substitution foralanine, mentioned in Example 5, is highlighted with an asterisk (*).

FIGS. 3A, B, and C shows the fatty acid profile of transgenic yeastexpressing the Calendula fatty acid-modifying enzyme associated withconjugated double bond formation comprising a modification of thedelta-9 position of fatty acids. Shown are gas chromatograms of fattyacid methyl esters prepared from (A) wild-type yeast, (B) transgenicyeast expressing the Calendula CalFad2-1 gene, and (C) from wild-typeCalendula seeds.

FIGS. 4A, B, and C shows the fatty acid profile of transgenic tobaccocallus expressing the Calendula fatty acid-modifying enzyme associatedwith conjugated double bond formation comprising a modification of thedelta-9 position of fatty acids. Shown are gas chromatograms of fattyacid methyl esters prepared from (A) wild-type tobacco callus, (B)transgenic tobacco callus expressing the Calendula CalFad2-1 gene, and(C) fatty acids isolated from wild-type Calendula seeds.

FIGS. 5A, B, and C shows a gas chromatographic analysis of fatty acidmethyl esters prepared from somatic soybean embryos expressingCalFad2-2. Shown are gas chromatograms of fatty acid methyl esters from(A) untransformed soybean embryos, (B) transgenic embryos expressingCalFad2-2, and (C) a standard fatty acid methyl ester mix prepared fromseeds of Punica granatum, Momordica charantia, and Calendulaofficinalis, all of which accumulate fatty acids with conjugated doublebonds. Punica seeds accumulate punicic acid(18:3Δ^(9cis,11trans,13cis)), Momordica seeds accumulate α-eleostearicacid (18:3Δ^(9cis,11trans,13trans)), and Calendula seeds accumulatecalendic acid (18:3Δ^(8trans,10trans,12cis)). As shown, the novel fattyacid methyl ester peak in soybean embryos expressing CalFad2-2 has thesame retention time (3.26 min) as methyl calendic acid from Calendulaofficinalis seeds. (The peak in B labeled with an asterisk istentatively identified as methyl 18:3Δ^(8trans, 10trans, 12trans)).

FIGS. 6A and B shows a mass spectral analysis of4-methyl-1,2,4-triazoline-3,5-dione (MTAD) derivatives of methylcalendic acid from (A) Calendula officinalis seeds and from (B)transgenic somatic soybean embryos expressing CalFad2-2. The MTADreagent preferentially reacts with the conjugated Δ^(8trans) andΔ^(10trans) double bonds of methyl calendic acid to yield the derivativeshown in A. As indicated, the mass spectrum of the MTAD derivativeprepared from transgenic soybean embryos expressing CalFad2-2 (B) isidentical to that of the MTAD derivative of methyl calendic acid fromCalendula seeds (A). A similar mass spectrum was also obtained from MTADderivatives prepared from transgenic soybean embryos expressingCalFad2-1 (data not shown).

FIG. 7 shows the biosynthesis of dimorphecolic acid in transgenicsomatic soybean embryos. The biosynthetic pathway is based on resultsfrom the transgenic expression of DMFad2-1 and DMFad2-2 as described inExample 11.

FIGS. 8A, B, and C shows the gas chromatographic analyses of fatty acidmethyl esters from (A) untransformed somatic soybean embryos, (B)somatic soybean embryos expressing DMFad2-1, and (C) developingDimorphotheca sinuata seeds. The peak labeled 18:2i corresponds to thetrans-Δ¹² isomer of linoleic acid (18:2Δ^(9cis,12trans)). The peaklabeled Dimorph. in panel C corresponds to dimorphecolic acid.

FIGS. 9A, B, and C shows the selected ion chromatograms from GC-MSanalyses of fatty acid methyl ester derivatives from (A) developingDimorphotheca sinuata seeds, (B) transgenic somatic soybean embryosexpressing DMFad2-2 and (C) transgenic somatic soybean embryosco-expressing DMFad2-1 and DMFad2-2. Chromatograms were obtained byscanning for the 225 m/z ion, which is the primary ion of the trimethylsilyl derivative of methyl dimorphecolic acid. Extracts from somaticsoybean embryos shown in panel B lacked detectable amounts of the18:2Δ^(9cis, 12trans), the preferred substrate for dimorphecolic acidsynthesis which is formed by the activity of DMFad2-1. In contrast,18:2Δ^(9cis, 12trans) composed >10% of the total fatty acids in extractsfrom somatic soybean embryos shown in panel C. cis-Dimorph.=thetentatively identified cis-Δ¹² isomer of dimorphecolic acid (9-OH-18:2Δ^(9cis, 12cis)). Dimorph.=dimorphecolic acid(9-OH-18:2Δ^(9cis, 12trans)).

FIGS. 10A and B shows the mass spectra of the trimethyl silyl derivativeof methyl dimorphecolic acid from developing Dimorphotheca sinuata seeds(A) and transgenic somatic soybean embryos co-expressing DMFad2-1 andDMFad2-2 (B).

SEQ ID NO:1 is the nucleotide sequence comprising the cDNA insert inclone ecs1c.pk009.n14 (CalFad2-1) encoding an fatty acid modifyingenzymes associated with conjugated double bond formation comprising amodification of the delta-9 position of fatty acids from seeds ofCalendula officinalis.

SEQ ID NO:2 is the deduced amino acid sequence of the nucleotidesequence comprising the cDNA insert in CalFad2-1.

SEQ ID NO:3 is the nucleotide sequence comprising the cDNA insert inclone ecs1c.pk008.a24 (CalFad2-2) encoding fatty acid modifying enzymesassociated with conjugated double bond formation comprising amodification of the delta-9 position of fatty acids from seeds ofCalendula officinalis.

SEQ ID NO:4 is the deduced amino acid sequence of the nucleotidesequence comprising the cDNA insert in CalFad2-2.

SEQ ID NO:5 is the amino acid sequence encoding the soybean (Glycinemax) fatty acid desaturase enzyme depicted in FIG. 2.

SEQ ID NO:6 is the amino acid sequence encoding the castor bean (Ricinuscommunis) fatty acid hydroxylase enzyme depicted in FIG. 2.

SEQ ID NO:7 is the deduced amino acid sequence of the nucleotidesequence comprising the cDNA insert in clone ImpH8Fad2 encoding an fattyacid modifying enzymes associated with conjugated double bond formationfrom seeds of Impatiens balsamina.

SEQ ID NO:8 is the deduced amino acid sequence of the nucleotidesequence comprising the cDNA insert in clone MomFad2 encoding fatty acidmodifying enzymes associated with conjugated double bond formation fromseeds of Momordica charantia.

SEQ ID NO:9 is the deduced amino acid sequence of the nucleotidesequence comprising the cDNA insert in the clone from ChrFad2 encoding afatty acid modifying enzymes associated with conjugated double bondformation from seeds of Chrysobalanus icaco.

SEQ ID NO:10 is the nucleotide sequence comprising the cDNA insert inclone dms2c.pk006.d7 (DMFad2-1) encoding an fatty acid modifying enzymesassociated with modification of the delta-12 position of fatty acidsfrom seeds of Dimorphotheca sinuata.

SEQ ID NO:11 is the deduced amino acid sequence of the nucleotidesequence comprising the cDNA insert in DMFad2-1.

SEQ ID NO:12 is the nucleotide sequence comprising the cDNA insert inclone dms2c.pk001.113 (DMFad2-2) encoding fatty acid modifying enzymesassociated with conjugated double bond formation comprising amodification of the delta-9 position of fatty acids from seeds ofDimorphotheca sinuata.

SEQ ID NO:13 is the deduced amino acid sequence of the nucleotidesequence comprising the cDNA insert in DMFad2-2.

SEQ ID NO:14 is the amino acid sequence encoding the sunflower(Helianthus annuus) fatty acid desaturase enzyme depicted in FIG. 2.

SEQ ID NO:15 is the amino acid sequence encoding the borage (Boragoofficinalis) fatty acid hydroxylase enzyme depicted in FIG. 2.

SEQ ID NO:16 is the BamHI-containing 5′-end “sense” primer used toamplify the Calendula officinalis coding region for cloning into thevector pBI121 for expression in tobacco.

SEQ ID NO:17 is the SstI-containing 3′-end “anti-sense” primer used toamplify the Calendula officinalis coding region for cloning into thevector pBI121 for expression in tobacco.

SEQ ID NO:18 is the NotI-containing 5′-end “sense” primer used toamplify the Calendula officinalis CalFad2-1 coding region for cloninginto the vector pKS67 for expression in soybean.

SEQ ID NO:19 is the NotI-containing 3′-end “anti-sense” primer used toamplify the Calendula officinalis CalFad2-1 coding region for cloninginto the vector pKS67 for expression in soybean.

SEQ ID NO:20 is the NotI-containing 5′-end “sense” primer used toamplify the Calendula officinalis CalFad2-2 coding region for cloninginto the vector pKS67 for expression in soybean.

SEQ ID NO:21 is the NotI-containing 3′-end “anti-sense” primer used toamplify the Calendula officinalis CalFad2-2 coding region for cloninginto the vector pKS67 for expression in soybean. SEQ ID NO:22 is theNotI-containing 5′-end “sense” primer used to amplify the Dimorphothecasinuata DMFad2-1 coding region for cloning into the vector pKS67 forexpression in soybean.

SEQ ID NO:23 is the NotI-containing 3′-end “anti-sense” primer used toamplify the Dimorphotheca sinuata DMFad2-1 coding region for cloninginto the vector pKS67 for expression in soybean.

SEQ ID NO:24 is the NotI-containing 5′-end “sense” primer used toamplify the Dimorphotheca sinuata DMFad2-2 coding region for cloninginto the vector pKS67 for expression in soybean.

SEQ ID NO:25 is the NotI-containing 3′-end “anti-sense” primer used toamplify the Dimorphotheca sinuata DMFad2-2 coding region for cloninginto the vector pKS67 for expression in soybean.

DETAILED DESCRIPTION OF THE INVENTION

In the context of this disclosure, a number of terms shall be utilized.

As used herein, an “isolated nucleic acid fragment” is a polymer of RNAor DNA that is single- or double-stranded, optionally containingsynthetic, non-natural or altered nucleotide bases. An isolated nucleicacid fragment in the form of a polymer of DNA may be comprised of one ormore segments of cDNA, genomic DNA or synthetic DNA.

The terms “subfragment that is functionally equivalent” and“functionally equivalent subfragment” are used interchangeably herein.These terms refer to a portion or subsequence of an isolated nucleicacid fragment in which the ability to alter gene expression or produce acertain phenotype is retained whether or not the fragment or subfragmentencodes an active enzyme. For example, the fragment or subfragment canbe used in the design of chimeric genes to produce the desired phenotypein a transformed plant. Chimeric genes can be designed for use inco-suppression or antisense by linking a nucleic acid fragment orsubfragment thereof, whether or not it encodes an active enzyme, in theappropriate orientation relative to a plant promoter sequence.

The terms “substantially similar” and “corresponding substantially” asused herein refer to nucleic acid fragments wherein changes in one ormore nucleotide bases does not affect the ability of the nucleic acidfragment to mediate gene expression or produce a certain phenotype.These terms also refer to modifications of the nucleic acid fragments ofthe instant invention such as deletion or insertion of one or morenucleotides that do not substantially alter the functional properties ofthe resulting nucleic acid fragment relative to the initial, unmodifiedfragment. It is therefore understood, as those skilled in the art willappreciate, that the invention encompasses more than the specificexemplary sequences.

Moreover, the skilled artisan recognizes that substantially similarnucleic acid sequences encompassed by this invention are also defined bytheir ability to hybridize, under moderately stringent conditions (forexample, 0.5×SSC, 0.1% SDS, 60° C.) with the sequences exemplifiedherein, or to any portion of the nucleotide sequences reported hereinand which are functionally equivalent to the promoter of the invention.Preferred substantially similar nucleic acid sequences encompassed bythis invention are those sequences that are 40% identical to the nucleicacid fragments reported herein or which are 40% identical to any portionof the nucleotide sequences reported herein. More preferred are nucleicacid fragments which are 50% identical to the nucleic acid sequencesreported herein, or which are 50% identical to any portion of thenucleotide sequences reported herein. Most preferred are nucleic acidfragments which are 60% identical to the nucleic acid sequences reportedherein, or which are 60% identical to any portion of the nucleotidesequences reported herein. Sequence alignments and percent similaritycalculations may be determined using the Megalign program of theLASARGENE bioinformatics computing suite (DNASTAR Inc., Madison, Wis.).Multiple alignment of the sequences are performed using the Clustalmethod of alignment (Higgins and Sharp (1989) CABIOS. 5:151-153) withthe default parameters (GAP PENALTY=10, GAP LENGTH PENALTY=10). Defaultparameters for pairwise alignments and calculation of percent identityof protein sequences using the Clustal method are KTUPLE=1, GAPPENALTY=3, WINDOW=5 and DIAGONALS SAVED=5. For nucleic acids theseparameters are KTUPLE=2, GAP PENALTY=5, WINDOW=4 and DIAGONALS SAVED=4.

A “substantial portion” of an amino acid or nucleotide sequencecomprises enough of the amino acid sequence of a polypeptide or thenucleotide sequence of a gene to afford putative identification of thatpolypeptide or gene, either by manual evaluation of the sequence by oneskilled in the art, or by computer-automated sequence comparison andidentification using algorithms such as BLAST (Altschul, S. F., et al.,(1993) J. Mol. Biol. 215:403-410) and Gapped Blast (Altschul, S. F. etal., (1997) Nucleic Acids Res. 25:3389-3402).

“Gene” refers to a nucleic acid fragment that expresses a specificprotein, including regulatory sequences preceding (5′ non-codingsequences) and following (3′ non-coding sequences) the coding sequence.“Native gene” refers to a gene as found in nature with its ownregulatory sequences. “Chimeric gene” refers any gene that is not anative gene, comprising regulatory and coding sequences that are notfound together in nature. Accordingly, a chimeric gene may compriseregulatory sequences and coding sequences that are derived fromdifferent sources, or regulatory sequences and coding sequences derivedfrom the same source, but arranged in a manner different than that foundin nature. “Endogenous gene” refers to a native gene in its naturallocation in the genome of an organism. A “foreign” gene refers to a genenot normally found in the host organism, but that is introduced into thehost organism by gene transfer. Foreign genes can comprise native genesinserted into a non-native organism, or chimeric genes. A “transgene” isa gene that has been introduced into the genome by a transformationprocedure.

“Coding sequence” refers to a DNA sequence that codes for a specificamino acid sequence. “Regulatory sequences” refer to nucleotidesequences located upstream (5′ non-coding sequences), within, ordownstream (3′ non-coding sequences) of a coding sequence, and whichinfluence the transcription, RNA processing or stability, or translationof the associated coding sequence. Regulatory sequences may include, butare not limited to, promoters, translation leader sequences, introns,and polyadenylation recognition sequences.

“Promoter” refers to a DNA sequence capable of controlling theexpression of a coding sequence or functional RNA. The promoter sequenceconsists of proximal and more distal upstream elements, with the latterelements often referred to as enhancers. Accordingly, an “enhancer” is aDNA sequence which can stimulate promoter activity and may be an innateelement of the promoter or a heterologous element inserted to enhancethe level or tissue-specificity of a promoter. Promoters may be derivedin their entirety from a native gene, or be composed of differentelements derived from different promoters found in nature, or evencomprise synthetic DNA segments. It is understood by those skilled inthe art that different promoters may direct the expression of a gene indifferent tissues or cell types, or at different stages of development,or in response to different environmental conditions. Promoters whichcause a gene to be expressed in most cell types at most times arecommonly referred to as “constitutive promoters”. New promoters ofvarious types useful in plant cells are constantly being discovered;numerous examples may be found in the compilation by Okamuro andGoldberg, (1989) Biochemistry of Plants 15:1-82. It is furtherrecognized that since in most cases the exact boundaries of regulatorysequences have not been completely defined, DNA fragments of somevariation may have identical promoter activity.

An “intron” is an intervening sequence in a gene that does not encode aportion of the protein sequence. Thus, such sequences are transcribedinto RNA but are then excised and are not translated. The term is alsoused for the excised RNA sequences. An “exon” is a portion of thesequence of a gene that is transcribed and is found in the maturemessenger RNA derived from the gene, but is not necessarily a part ofthe sequence that encodes the final gene product.

The “translation leader sequence” refers to a DNA sequence locatedbetween the promoter sequence of a gene and the coding sequence. Thetranslation leader sequence is present in the fully processed mRNAupstream of the translation start sequence. The translation leadersequence may affect processing of the primary transcript to mRNA, mRNAstability or translation efficiency. Examples of translation leadersequences have been described (Turner, R. and Foster, G. D. (1995)Molecular Biotechnology 3:225).

The “3′ non-coding sequences” refer to DNA sequences located downstreamof a coding sequence and include polyadenylation recognition sequencesand other sequences encoding regulatory signals capable of affectingmRNA processing or gene expression. The polyadenylation signal isusually characterized by affecting the addition of polyadenylic acidtracts to the 3′ end of the mRNA precursor. The use of different 3′non-coding sequences is exemplified by Ingelbrecht et al., (1989) PlantCell 1:671-680.

“RNA transcript” refers to the product resulting from RNApolymerase-catalyzed transcription of a DNA sequence. When the RNAtranscript is a perfect complementary copy of the DNA sequence, it isreferred to as the primary transcript or it may be a RNA sequencederived from posttranscriptional processing of the primary transcriptand is referred to as the mature RNA. “Messenger RNA (mRNA)” refers tothe RNA that is without introns and that can be translated into proteinby the cell. “cDNA” refers to a DNA that is complementary to andsynthesized from a mRNA template using the enzyme reverse transcriptase.The cDNA can be single-stranded or converted into the double-strandedform using the klenow fragment of DNA polymerase I. “Sense” RNA refersto RNA transcript: that includes the mRNA and so can be translated intoprotein within a cell or in vitro. “Antisense RNA” refers to a RNAtranscript that is complementary to all or part of a target primarytranscript or mRNA and that blocks the expression of a target gene (U.S.Pat. No. 5,107,065). The complementarity of an antisense RNA may be withany part of the specific gene transcript, i.e., at the 5′ non-codingsequence, 3′ non-coding sequence, introns, or the coding sequence.“Functional RNA” refers to antisense RNA, ribozyme RNA, or other RNAthat may not be translated but has an effect on cellular processes. Theterms “complement” and “reverse complement” are used interchangeablyherein with respect to mRNA transcripts, and are meant to define theantisense RNA of the message.

The term “operably linked” refers to the association of nucleic acidsequences on a single nucleic acid fragment so that the function of oneis affected by the other. For example, a promoter is operably linkedwith a coding sequence when it is capable of affecting the expression ofthat coding sequence (i.e., that the coding sequence is under thetranscriptional control of the promoter). Coding sequences can beoperably linked to regulatory sequences in sense or antisenseorientation.

The term “expression”, as used herein, refers to the production of afunctional end-product. Expression or overexpression of a gene involvestranscription of the gene and translation of the mRNA into a precursoror mature protein. “Antisense inhibition” refers to the production ofantisense RNA transcripts capable of suppressing the expression of thetarget protein. “Overexpression” refers to the production of a geneproduct in transgenic organisms that exceeds levels of production innormal or non-transformed organisms. “Co-suppression” refers to theproduction of sense RNA transcripts capable of suppressing theexpression of identical or substantially similar foreign or endogenousgenes (U.S. Pat. No. 5,231,020).

“Altered expression” refers to the production of gene product(s) intransgenic organisms in amounts or proportions that differ significantlyfrom that activity in comparable tissue (organ and of developmentaltype) from wild-type organisms.

“Mature” protein refers to a post-translationally processed polypeptide;i.e., one from which any pre- or propeptides present in the primarytranslation product have been removed. “Precursor” protein refers to theprimary product of translation of mRNA; i.e., with pre- and propeptidesstill present. Pre- and propeptides may be but are not limited tointracellular localization signals.

A “chloroplast transit peptide” is an amino acid sequence which istranslated in conjunction with a protein and directs the protein to thechloroplast or other plastid types present in the cell in which theprotein is made. “Chloroplast transit sequence” refers to a nucleotidesequence that encodes a chloroplast transit peptide. A “signal peptide”is an amino acid sequence which is translated in conjunction with aprotein and directs the protein to the secretory system (Chrispeels, J.J., (1991) Ann. Rev. Plant Phys. Plant Mol. Biol. 42:21-53). If theprotein is to be directed to a vacuole, a vacuolar targeting signal(supra) can further be added, or if to the endoplasmic reticulum, anendoplasmic reticulum retention signal (supra) may be added. If theprotein is to be directed to the nucleus, any signal peptide presentshould be removed and instead a nuclear localization signal included(Raikhel (1992) Plant Phys. 100:1627-1632).

“Transformation” refers to the transfer of a nucleic acid fragment intothe genome of a host organism, resulting in genetically stableinheritance. Host organisms containing the transformed nucleic acidfragments are referred to as “transgenic” organisms. The preferredmethod of cell transformation of rice, corn and other monocots is theuse of particle-accelerated or “gene gun” transformation technology(Klein et al., (1987) Nature (London) 327:70-73; U.S. Pat. No.4,945,050), or an Agrobacterium-mediated method using an appropriate Tiplasmid containing the transgene (Ishida Y. et al., 1996, NatureBiotech. 14:745-750).

Standard recombinant DNA and molecular cloning techniques used hereinare well known in the art and are described more fully in Sambrook, J.,Fritsch, E. F. and Maniatis, T. Molecular Cloning: A Laboratory Manual;Cold Spring Harbor Laboratory Press: Cold Spring Harbor, 1989(hereinafter “Sambrook”).

“PCR” or “Polymerase Chain Reaction” is a technique for the synthesis oflarge quantities of specific DNA segments, consists of a series ofrepetitive cycles (Perkin Elmer, Cetus Instruments, Norwalk, Conn.).Typically, the double stranded DNA is heat denatured, the two primerscomplementary to the 3′ boundaries of the target segment are annealed atlow temperature and then extended at an intermediate temperature. Oneset of these three consecutive steps is referred to as a cycle.

An “expression construct” as used herein comprises any of the isolatednucleic acid fragments of the invention used either alone or incombination with each other as discussed herein and further may be usedin conjunction with a vector or a subfragment thereof. If a vector isused then the choice of vector is dependent upon the method that will beused to transform host plants as is well known to those skilled in theart. For example, a plasmid vector can be used. The skilled artisan iswell aware of the genetic elements that must be present on the vector inorder to successfully transform, select and propagate host cells orplants comprising any of the isolated nucleic acid fragments of theinvention. The skilled, artisan will also recognize that differentindependent transformation events will result in different levels andpatterns of expression (Jones et al., (1985) EMBO J. 4:2411-2418; DeAlmeida et al., (1989) Mol. Gen. Genetics 218:78-86), and thus thatmultiple events must be screened in order to obtain lines displaying thedesired expression level and pattern. Such screening may be accomplishedby Southern analysis of DNA, Northern analysis of mRNA expression,Western analysis of protein expression, or phenotypic analysis. Theterms “expression construct” and “recombinant expression construct” areused interchangeably herein.

The term “ω⁶-oleic acid desaturase” refers to a cytosolic enzyme thatcatalyzes the insertion of a double bond into oleic acid between thetwelfth and thirteenth carbon atoms relative to the carboxyl end of theacyl chain. Double bonds are referred to as “cis” or “trans” becausethey are chiral units that can assume the following non-equivalentstructures:

The linoleic acid substrate for this enzyme may be bound to aglycerolipid such as phosphatidylcholine. In fatty acid chains theomega-carbons are counted from the methyl-end, while the delta-carbonsare counted from the carboxyl-end. Thus, the term “delta-9 position”, asused herein means the 9th carbon atom counting from the carboxyl-end ofthe fatty acid chain. Modifications involving the delta-9 positioninclude, but are not limited to, at least one modification selected fromthe group consisting of double bond formation, conjugated double bondformation, hydroxylation, epoxidation, hydroxy-conjugation, and thelike. For example, a modification can involve just one alteration suchas conjugated double bond formation or a modification can involve morethan one alteration such as conjugated double bond formation andhydroxylation (hydroxy-conjugation). The term “modification of thedelta-9 (Δ⁹) position” and “a modified delta-9 (Δ⁹) position” are usedinterchangeably. Also, the term “modification of the delta-12 position”,as used herein means a double bond formation involving the 12^(th)carbon counting from the carboxyl-end of the fatty acid chain. Thismodification as described in the present invention involves theformation of a trans-Δ¹² double bond resulting in the formation oftrans-linoleic acid (18:2Δ^(9cis, 12trans)).

In the production of calendic acid, the delta-9 double bond of linoleicacid (18:2Δ^(9,12)) is converted by the activity of CalFad2-1 orCalFad2-2 to delta-8 and delta-10 double bonds. The resulting calendicacid, a linolenic acid derivative, contains delta-8, delta-10, anddelta-12 double bonds in conjugation (18:3Δ^(8,10,12)). CalFad2-1 andCalFad2-2 are thus distinct from all previously reported Fad2-relatedpolypeptides by their ability to modify the delta-9 rather than thedelta-12 position of a fatty acid. The enzymes from Impatiens balsamina,Momordica charantia, and Chrysobalanus icaco, shown in FIG. 2, allconvert the delta-12 double bond of linoleic acid to delta-11 anddelta-13 conjugated double bonds, to form eleostearic acid(18:3Δ^(9,11,13)).

In the production of dimorphecolic acid(9-hydroxy-18:2Δ^(10trans, 12trans), see FIG. 7 for the structure) oleicacid is first converted to trans-linoleic acid (18:2Δ^(9cis, 12trans))by the enzyme designated DMFad2-1. This enzyme (DMFad2-1) is aDimorphotheca sinuata fatty acid modifying enzyme associated with theformation of a trans delta-12 double bond wherein said enzyme modifies adelta-12 position of fatty acids by inserting a double bond having atrans configuation between carbon atoms 12 and 13. The resulting productof this enzymatic reaction is trans-linoleic acid. This product thenbecomes the substrate for the next enzymatic reaction in this pathway.Specifically, trans-linoleic acid is converted by the enzyme DMFad2-2 todimorphecolic acid, which is a conjugated double-bond containing fattyacid. The enzyme DMFad2-2 is a Δ-9 hydroxy-conjugase from Dimorphothecasinuata. The enzyme introduces a hydroxyl group at position 9 andconverts 18:2Δ^(9cis, 12trans) (trans-linoleic acid) to the conjugateddouble bond containing dimorphecolic acid(9-hydroxy-18:2Δ^(10trans, 12trans)). A related product,cis-dimorphecolic acid (9-hydroxy -18:2Δ^(10trans, 12cis), see FIG. 7for the structure) is produced by DMFad2-2 from endogenous soybeanlinoleic acid (18:2Δ^(9trans, 12cis)). It is believed that the trans-form of linoleic acid is the preferred substrate for DMFad2-2.

The enzymes of the present invention, with the exception of DMFad2-1,comprise activities involving modification of fatty acids at the delta-9position resulting in conjugated double bond formation. The term“conjugated double bond” is defined as two double bonds in the relativepositions indicated by the formula —CH═CH—CH═CH— (Grant & Hackh'sChemical Dictionary, Fifth Ed., R. Grant and C. Grant eds., McGraw-Hill,New York). The π-orbital electrons are shared between conjugated doublebonds, but remain relatively independent in unconjugated double bonds.This explains the greater reactivity of conjugated double bonds tooxidation. The modifying enzymes, associated with conjugated double bondformation described herein, are related to, and share sequence homologyto, the fatty acid desaturases (Fads), especially the Fad2 class. Fadsintroduce double bonds in fatty acid chains that result in the formationof the mono and polyunsaturated oils, such as oleate,: linoleate, andlinolenate, but do not produce conjugated double bonds. The terms “Fad2related” and “Fad2-like” reflect the conservation and differences innucleic acid sequence homology between the genes encoding Fad2 enzymesversus the genes of the present invention.

This invention concerns an isolated nucleic acid fragment encoding aplant fatty acid modifying enzyme associated with conjugated double bondformation comprising the delta-9 position of fatty acids, or in the caseof DMFad2-1, modification of a delta-12 position, wherein said fragmentor a functionally equivalent subfragment thereof (a) hybridizes to anyof the nucleotide sequences set forth in SEQ ID NOs:1, 3, or 12 underconditions of moderate stringency or (b) is at least 40% identical to apolypeptide encoded by any of the nucleotide sequences set forth in SEQID NOs:1, 3, or 12 or a functionally equivalent subfragment thereof asdetermined by a comparison method designed to detect homologoussequences.

This invention also concerns an isolated nucleic acid fragment encodinga plant fatty acid modifying enzyme wherein said enzyme modifies adelta-9 position of fatty acids and further wherein said fragment or afunctionally equivalent subfragment thereof (a) hybridizes to any of thenucleotide sequences set forth in SEQ ID NOs:1, 3, or 12 underconditions of moderate stringency or (b) is at least 40% identical to apolypeptide encoded: by any of the nucleotide sequences set forth in SEQID NOs:1, 3, or 12 or a functionally equivalent subfragment thereof asdetermined by a comparison method designed to detect homologoussequences.

Such enzymes are normally expressed in developing seeds of Calendulaofficinalis or Dimorphotheca sinuata that are similar in sequence toplant, membrane-bound fatty acid desaturases. However, these fatty acidmodifying enzymes differ from membrane-bound fatty acid desaturases intheir functionality. Specifically, these enzymes are associated with theformation of fatty acids having conjugated double bonds and, moreparticularly, with the formation of conjugated linolenic acids. Examplesof fatty acids having conjugated double bonds include, but are notlimited to, eleostearic acid and/or parinaric acid. Naturally occurringplant oils containing eleostearic acid include tung oil from Aleuritesfordii or montana, which contains up to 69% α-eleostearic acid in theoil extracted from the seeds, or oils from valarian species (Centranthusmicrosiphon). There can also be mentioned jacaric acid (from thejacaranda tree, Jacaranda mimosifolia and Jacaranda chelonia,18:3Δ^(8cis, 10trans, 12cis)), calendic acid (from marigold or Africandaisy, Calendula officinalis, and Osteospermum spinescens andOsteospermum hyoseroides, 18:3Δ^(8trans, 10trans, 12cis)), catalpic acid(from the trumpet creeper, Catalpa ovata, or speciosa, or bigninioides,18:3Δ^(9trans, 11trans, 13cis)), and punicic acid (from bitter melon andpomegranate, or Tricosanthes species, Cucurbita, and Punica granatum,Tricosanthes cucumeroides, 18:3Δ^(9cis, 11trans, 13cis)). These andother examples of fatty acids having conjugated double bonds may befound in “The Lipid Handbook” (Second Edition, Gunstone, F. D., et al.,eds., Chapman and Hall, London, 1994), Crombie and Holloway (J. Chem.Soc. Perkins Trans. 1985:2425-2434), and Liu, et al. (Plant. Physiol.[1997] 113:1343-1349). These conjugated fatty acids are also referred toas ClnAs (conjugated linolenic acids) because they are all 18:3 incomposition. This is in contrast to CLAs (conjugated linoleic acids)which have an 18:2 configuration.

The nomenclature “18:3” denotes the number of carbons in the fatty acidchain (in this case “18” or stearic acid length), and the number ofunsaturating double bonds (in this case “3” specifying this fatty acidas linolenic). Although 18:2 and 18:3 denote linoleic acid and linolenicacid, respectively, the positions of the double bonds are not specified(i.e. they may be unconjugated or conjugated, cis or trans). The term“calendic acid” as used herein refers to a mixture of cis-trans isomersof Δ^(8,10,12)-octadecatrienoic acid (18:3Δ^(8,10,12)). This mixture isprimarily composed of the Δ^(8trans, 10trans, 12cis) isomer ofoctadecatrienoic acid (18:3) but may also contain various cis-transisomers of this fatty acid. As those skilled in the will appreciate, thevarious isomers of calendic acid are separated easily by gaschromatography-mass spectrometry (GC-MS, see FIG. 3). More details onGC-MS analyses are found in Examples 3, 4, 6, 7, and 8. The term“dimorphecolic acid” as used herein refers to9-hydroxy-18:2Δ^(10trans, 12trans) (see FIG. 7 for the structure). Thisunusual fatty acid and the intermediate that is its precursor(trans-linoleic acid, 18:2Δ^(9cis, 12trans)) can be analyzed by GC-MSanalyses (see Example 11) and by ¹H-¹³C NMR two-dimensional correlationNMR (see Example 12).

Examples of comparison methods which detect sequence homology includebut are not limited to the BLAST computational method (Basic LocalAlignment Search Tool; Altschul et al. (1993) J. Mol. Biol. 215:403-410)which includes BLASTN (nucleotide, both strands), BLASTX (nucleotide,six-frame translation), BLASTP (protein), TBLASTN (protein, fromsix-frame translation), TBLASTX (nucleotide, six-frame translation),Megalign program of the LASARGENE bioinformatics computing suite(DNASTAR Inc., Madison, Wis., used for calculating percent identity),and the Clustal method of multiple sequence alignment (Higgins and Sharp(1989) CABIOS. 5:151-153). The default parameters were used for allcomparisons and for all methods. The BLAST suite at NCBI has a detaileddiscussion of their algorithms at their web site, the Megalign programuses a Clustal program that shares default parameters with Clustal,namely, for multiple sequence alignments of nucleic acids orpolypeptides (GAP PENALTY=10, GAP LENGTH PENALTY=10), for pairwisealignments of nucleic acids (KTUPLE=2, GAP PENALTY=5, WINDOW=4,DIAGONALS SAVED=4), and for pairwise alignments of polypeptides(KTUPLE=1, GAP PENALTY=3, WINDOW=5, DIAGONALS SAVED=5).

This invention also relates to the following:

a) an isolated nucleic acid fragment encoding a plant fatty acidmodifying enzyme associated with conjugated double bond formationcomprising the delta-9 position of fatty acids wherein said fragmentencodes a protein comprising any one of the amino acid sequences setforth in SEQ ID NOs:2, 4, or 13, as well as

b) an isolated nucleic acid fragment encoding a plant fatty acidmodifying enzyme wherein said enzyme modifies a delta-9 position offatty acids and further wherein said fragment or a functionallyequivalent subfragment thereof encodes a protein comprising any one ofthe amino acid sequences set forth in SEQ ID NOs:2, 4, or 13.

In another aspect, this invention concerns an isolated nucleic acidfragment encoding a plant fatty acid modifying enzyme associated withconjugated double bond formation comprising a the delta-9 position offatty acids or an isolated nucleic acid fragment encoding a plant fattyacid modifying enzyme wherein said enzyme modifies the delta-9 positionof the fatty wherein said fragments or a functionally equivalentsubfragments thereof hybridize to any of the isolated nucleic acidfragments or functionally equivalent subfragments thereof encoding aplant fatty acid modifying enzyme associated with conjugated double bondformation comprising the delta-9 position of fatty acids or associatedwith modification of the delta-9 position wherein said fragments orsubfragments encode a protein comprising any one of the amino acidsequences set forth in SEQ ID NOs:2, 4, or 13 and further wherein saidfragments or subfragments (a) hybridize to these isolated nucleic acidfragments or functionally equivalent subfragments under conditions ofmoderate stringency or (b) is at least 40% identical to a polypeptideencoded by any of the foregoing isolated nucleic acid fragments or afunctionally equivalent subfragments thereof as determined by acomparison method designed to detect homologous sequences. Examples ofsuitable comparison methods which detect homologous sequences arediscussed above.

Also of interest is a chimeric gene comprising any of the instantisolated nucleic acid fragments, or functionally equivalent subfragmentsthereof, or a complement thereof operably linked to suitable regulatorysequences wherein expression of the chimeric gene results in productionof altered levels of the desired enzyme in a transformed host cell orplant.

The invention also relates to methods of using such isolated nucleicacid fragments, or functionally equivalent subfragments thereof, or thecomplement thereof, to alter the level of fatty acids comprising amodification of the delta-9 position of fatty acids in a host cell orplant which comprises:

(a) transforming a host cell or plant with any of the instant chimericgenes;

(b) growing the transformed host cell or plant under conditions suitablefor the expression of the chimeric gene; and

(c) selecting those transformed host cells or plants having alteredlevels of fatty acids comprising a modification at the delta-9 position.

In still another aspect, this invention concerns a method for producingseed oil containing fatty acids comprising a modification at the delta-9position in the seeds of plants which comprises:

(a) transforming a plant cell with any of the instant chimeric genes;

(b) growing a fertile mature plant from the transformed plant cell ofstep (a);

(c) screening progeny seeds from the fertile plants of step (b) foraltered levels of fatty acids comprising a modification at the delta-9position; and

(d) processing the progeny seed of step (c) to obtain seed oilcontaining altered levels plant fatty acids comprising a modification atthe delta-9 position.

In still a further aspect, this invention concerns a method forproducing plant fatty acid modifying enzymes associated withmodification of the delta-9 position of fatty acids which comprises:

(a) transforming a microbial host cell with any of the instant chimericgenes;

(b) growing the transformed host cell under conditions suitable for theexpression of the chimeric gene; and

(c) selecting those transformed host cells containing altered levels ofprotein encoded by the chimeric gene.

The isolated nucleic acid fragments encoding fatty acid modifyingenzymes associated with conjugated double bond formation comprising thedelta-9 position of fatty acids in seeds of Calendula officinalis isprovided in SEQ ID NO:1 and 3, and the corresponding deduced amino acidsequences are provided in SEQ ID NO:2 and 4, and in the seeds ofDimorphotheca sinuata is provided in SEQ ID NO:12, and the correspondingdeduced amino acid sequences are provided in SEQ ID NO:13. Fatty acidmodifying enzymes associated with conjugated double bond formationcomprising modification of the delta-9 position of fatty acids fromother plants fatty acid modifying enzymes which are capable of modifyingthe delta-9 position of a fatty acid can now be identified by whennucleotide sequence hybridizes to any of the nucleotide sequences setforth in SEQ ID NOS:1, 3, and 12 under conditions of moderatestringency, as set forth above, or (b) is at least 40% identical to apolypeptide encoded by any of the nucleotide sequences set forth in SEQID NOs:1, 3, or 12 or a functionally equivalent subfragment thereof asdetermined by a comparison method designed to detect homologoussequences.

The amino acid sequences encoded by these nucleotide sequences disclosedherein are compared in FIG. 2 to the sequences encoding enzymes involvedin conjugated fatty acid synthesis in Impatiens, Momordica,Chrysobalanus, and delta-12 desaturases from Helianthus and Borago, aswell as the fatty acid desaturases from soybean which inserts the seconddouble bond between carbon atoms 12 and 13 into monounsaturated fattyacid, oleic acid to produce linoleic acid.

The isolated nucleic acid fragments of the instant invention, orfunctionally equivalent subfragments thereof, or the complement thereof,can be used to create chimeric genes to transform host cells or plants.Examples of host cells which can be transformed include prokaryotic andeukaryotic cells. There can be mentioned microorganisms such as thebacterium E. coli and yeast Saccharomyces cerevisiae. Examples of plantcells include but are not limited to those obtained from soybean,oilseed Brassica species, corn, peanut, rice, wheat, sunflower,safflower, cotton, palm, flax, and cocoa.

Thus, the chimeric genes of the instant invention can be used to createtransgenic plants in which the fatty acid modifying enzymes which modifythe delta-9 position of fatty acids in seeds of Calendula officinalis orDimorphotheca sinuata are present at higher levels than normal or incell types or developmental stages in which it is not normally found.Also of interest are seeds obtained from such plants and oil obtainedfrom these seeds.

Transgenic plants can be made in which fatty acid modifying enzymeassociated with modification of the delta-9 position of fatty acids ispresent at lower levels than normal or in cell types or developmentalstages in which it is not normally found. This would have the effect ofaltering the level of such fatty acids comprising a modified delta-9position in those cells. It may be desirable to reduce or eliminateexpression of a gene encoding such enzymes in plants for someapplications. In order to accomplish this, a chimeric gene designed forco-suppression of the endogenous enzyme can be constructed by linking agene or gene fragment encoding a fatty acid modifying enzyme associatedwith modification of the delta-9 position of fatty acids to plantpromoter sequences. Alternatively, a chimeric gene designed to expressantisense RNA for all or part of the instant nucleic acid fragment canbe constructed by linking the gene or a gene fragment in reverseorientation to plant promoter sequences. Either the co-suppression orantisense chimeric genes could be introduced into plants viatransformation wherein expression of the corresponding endogenous genesare reduced or eliminated.

When over-expressed in plant cells, the fatty acid modifying enzymesassociated with modification of the delta-9 position of fatty acids inseeds of Calendula officinalis or Dimorphotheca sinuata can be usefulfor causing the biosynthesis and accumulation of fatty acids withconjugated double bonds, such as calendic acid, in those cells. It isparticularly useful to use fatty acid modifying enzymes associated withmodification of the delta-9 position of fatty acids in seeds ofCalendula officinalis or Dimorphotheca sinuata to produce fatty acidscontaining conjugated double bonds in the cells of the seeds of oilseedcrop plants.

Overexpression of fatty acid modifying enzymes associated withmodification of the delta-9 position of fatty acids in seeds ofCalendula officinalis or Dimorphotheca sinuata may be accomplished byfirst constructing a chimeric gene in which the coding region of cDNAsfor fatty acid modifying enzymes associated with modification of thedelta-9 position of fatty acids in seeds of Calendula officinalis orDimorphotheca sinuata is operably linked to a promoter capable ofdirecting expression of a gene in the desired tissues at the desiredstage of development. For reasons of convenience, the chimeric gene maycomprise a promoter sequence and translation leader sequence derivedfrom the same gene. 3′ non-coding sequences encoding transcriptiontermination signals must also be provided. The instant chimeric genesmay also comprise one or more introns in order to facilitate geneexpression.

Vectors, such as plasmid vectors, comprising the instant chimeric genescan then be constructed. The choice of plasmid vector is dependent uponthe method that will be used to transform host plants. The skilledartisan is well aware of the genetic elements that must be present onthe plasmid vector in order to successfully transform, select andpropagate host cells or plants containing the chimeric gene. The skilledartisan will also recognize that different independent transformationevents will result in different levels and patterns of expression (Joneset al., (1985) EMBO J. 4:2411-2418; De Almeida et al., (1989) Mol. Gen.Genetics 218:78-86), and thus that multiple events must be screened inorder to obtain lines displaying the desired expression level andpattern. Such screening may be accomplished by Southern analysis of DNA,Northern analysis of mRNA expression, Western analysis of proteinexpression, or phenotypic analysis.

For some applications it may be useful to direct the instant fatty acidmodifying enzymes associated with modification of the delta-9 positionof fatty acids in seeds of Calendula officinalis or Dimorphothecasinuata to different cellular compartments, or to facilitate itssecretion from the cell. It is thus envisioned that the chimeric genesdescribed above may be further supplemented by altering the codingsequences to encode fatty acid modifying enzymes associated withmodification of the delta-9 position of fatty acids in seeds ofCalendula officinalis or Dimorphotheca sinuata disclosed herein withappropriate intracellular targeting sequences such as transit sequences(Keegstra, K. (1989) Cell 56:247-253), signal sequences or sequencesencoding endoplasmic reticulum localization (Chrispeels, J. J., (1991)Ann. Rev. Plant Phys. Plant Mol. Biol. 42:21-53), or nuclearlocalization signals (Raikhel, N. (1992) Plant Phys. 100:1627-1632)added and/or with targeting sequences that are already present removed.While the references cited give examples of each of these, the list isnot exhaustive and more targeting signals of utility may be discoveredin the future.

The nucleic acid fragments of the instant invention, or functionallyequivalent subfragment thereof, may be used to isolate cDNAs and othernucleic acid fragments encoding homologous fatty acid modifying enzymesfrom the same or other plant species. Isolation of homologous genesusing sequence-dependent protocols is well known in the art. Examples ofsequence-dependent protocols include, but are not limited to, methods ofnucleic acid hybridization, and methods of DNA and RNA amplification asexemplified by various uses of nucleic acid amplification technologies(e.g., polymerase chain reaction, ligase chain reaction). The term“conserved sequence(s)” as used herein encompasses both strictconservation as well as conservation of a majority of the sequences usedin an alignment, for example, conservation with respect to a consensussequence.

Thus, in still a further aspect this invention concerns a method toisolate nucleic acid fragments and functionally equivalent subfragmentsthereof encoding a plant fatty acid modifying enzyme associated withmodification of the delta-9 position of fatty acids comprising:

(a) comparing SEQ ID NOs:2, 4, or 13 and other plant fatty acidmodifying enzyme polypeptide sequences;

(b) identifying conserved sequences of 4 or more amino acids obtained instep (a);

(c) designing degenerate oligomers based on the conserved sequencesidentified in step (b); and

(d) using the degenerate oligomers of step (s) to isolate sequencesencoding a plant fatty acid modifying enzyme or a portion thereofassociated with modification of the delta-9 position of fatty acids bysequence dependent protocols.

For example, genes encoding homologous fatty acid modifying enzymes,either as cDNAs or genomic DNAs, could be isolated directly by using allor a portion of the instant nucleic acid fragments as DNA hybridizationprobes to screen libraries from any desired plant employing methodologywell known to those skilled in the art. Specific oligonucleotide probesbased upon the instant nucleic acid sequences can be designed andsynthesized by methods known in the art (Sambrook). Moreover, the entiresequences can be used directly to synthesize DNA probes by methods knownto the skilled artisan such as random primers DNA labeling, nicktranslation, or end-labeling techniques, or RNA probes using availablein vitro transcription systems. In addition, specific primers can bedesigned and used to amplify a part of or full-length of the instantsequences. The resulting amplification products can be labeled directlyduring amplification reactions or labeled after amplification reactions,and used as probes to isolate full length cDNA or genomic fragmentsunder conditions of appropriate stringency.

In addition, two short segments of the instant nucleic acid fragmentsmay be used in polymerase chain reaction protocols to amplify longernucleic acid fragments encoding homologous genes from DNA or RNA. Thepolymerase chain reaction may also be performed on a library of clonednucleic acid fragments wherein the sequence of one primer is derivedfrom the instant nucleic acid fragments, and the sequence of the otherprimer takes advantage of the presence of the polyadenylic acid tractsto the 3′ end of the mRNA precursor encoding plant genes. Alternatively,the second primer sequence may be based upon sequences derived from thecloning vector. For example, the skilled artisan can follow the RACEprotocol (Frohman et al., (1988) PNAS USA 85:8998) to generate cDNAs byusing PCR to amplify copies of the region between a single point in thetranscript and the 3′ or 5′ end. Primers oriented in the 3′ and 5′directions can be designed from the instant sequences. Usingcommercially available 3′ RACE or 5′ RACE systems (BRL), specific 3′ or5′ cDNA fragments can be isolated (Qhara et al., (1989) PNAS USA86:5673; Loh et al., (1989) Science 243:217). Products generated by the3′ and 5′ RACE procedures can be combined to generate full-length cDNAs(Frohman, M. A. and Martin, G. R., (1989) Techniques 1:165).

Thus, other nucleic acid fragments encoding enzymes associated withmodification of the delta-9 position of fatty acids can be identifiedusing any of the general methodologies described above. For example, ageneral group of fatty acid desaturase (FAD) related cDNAs, can beidentified and a specific subset of those cDNAs encoding enzymesinvolved in modification of the delta-9 position of fatty acids can bedetected or screened by transformation. A group of cDNA sequencesencoding fatty acid desaturase-like enzymes can be identified usinglow-stringency hybridization (for example 2×SSC, 0.1% SDS, 60° C.) witha probe corresponding to any known FAD sequence, and/or all-or-part ofthe sequences presented in any of SEQ ID NOs:1, 3, or 12. Alternatively,randomly sequenced cDNAs can be analyzed by a computer program designedto detect homologous sequences, such as, but not limited to, BLAST orgapped BLAST (using standard default parameters). BLAST (Basic LocalAlignment Search Tool; Altschul et al. (1993) J. Mol. Biol. 215:403-410)searches for similarity to sequences contained in the BLAST “nr”database (comprising all non-redundant GenBank CDS translations,sequences derived from the 3-dimensional structure Brookhaven ProteinData Bank, the last major release of the SWISS-PROT protein sequencedatabase, EMBL, and DDBJ databases). Test sequences are analyzed forsimilarity to all publicly available DNA sequences contained in the “nr”database using the BLASTN algorithm provided by the National Center forBiotechnology Information (NCBI). The DNA sequences are translated inall reading frames and compared for similarity to all publicly availableprotein sequences contained in the “nr” database using the BLASTXalgorithm (Gish and States (1993) Nature Genetics 3:266-272) provided bythe NCBI. For convenience, the P-value (probability), or “pLog” (thenegative of the logarithm of the P-value), is given as a measure ofsimilarity between the two sequences. A test sequence and a sequencecontained in the searched databases are compared, and the probabilitythat the two sequences are related only by chance is calculated by BLASTand reported as a “pLog” value. Accordingly, the greater the pLog value,the greater the likelihood that the cDNA sequence and the BLAST “hit”represent homologous proteins. Sequences with pLogs greater than 5, orpreferably greater than 10, or more preferably greater than 15, and mostpreferably greater than 20, that are defined as FADs or lipiddesaturases are candidates. cDNAs encoding enzymes associated withmodification of the delta-9 position of fatty acids can be identifiedfrom the candidate pools using transformation screening. IndividualcDNAs are inserted into expression vectors and transformed into yeast orplant host cells using methods well known to those skilled in the art(see Examples 3, 4, 6, 7, and 8). Production of fatty acids containingconjugated double bonds is confirmed by GC-MS analyses as described inthe Examples 3 and 4. Yeast or plant tissue culture cells are preferredfor initial screening due to speed and the ease with which they can behandled when dealing with large numbers of transformants and theappropriate cell biology and eukaryotic cell physiology.

The instant fatty acid modifying enzymes associated with modification ofthe delta-9 position of fatty acids in seeds of Calendula officinalis orDimorphotheca sinuata produced in heterologous host cells or plants,particularly in the cells of microbial hosts, can be used to prepareantibodies to the fatty acid modifying enzymes associated withmodification of the delta-9 position of fatty acids in seeds ofCalendula officinalis or Dimorphotheca sinuata by methods well known tothose skilled in the art. The antibodies are useful for detecting theinstant fatty acid modifying enzymes associated with modification of thedelta-9 position of fatty acids in seeds of Calendula officinalis orDimorphotheca sinuata in situ in cells or in vitro in cell extracts.Preferred heterologous host cells for production of the instant fattyacid modifying enzymes associated with modification of the delta-9position of fatty acids in seeds of Calendula officinalis orDimorphotheca sinuata are microbial hosts. Microbial expression systemsand expression vectors containing regulatory sequences that direct highlevel expression of foreign proteins are well known to those skilled inthe art. Any of these could be used to construct chimeric genes forproduction of the instant fatty acid modifying enzymes associated withmodification of the delta-9 position of fatty acids in seeds ofCalendula officinalis or Dimorphotheca sinuata. These chimeric genescould then be introduced into appropriate microorganisms viatransformation to provide high level expression of the encoded fattyacid modifying enzymes associated with modification of the delta-9position of a fatty acid in seeds Calendula officinalis or Dimorphothecasinuata. An example of the use of the Calendula officinalis orDimorphotheca sinuata fatty acid modifying enzyme in Saccharomycescerevisiae for the production of calendic acid is discussed below inExample 4. An example of a vector for high level expression of theinstant fatty acid modifying enzymes associated with modification of thedelta-9 position of fatty acids in seeds of Calendula officinalis orDimorphotheca sinuata in a bacterial host is discussed below in Example8.

In still another aspect, it has been found that fatty acids modified atthe delta-9 position, and in particular, those fatty acids havingconjugated double bonds comprising the delta-9 position, morespecifically, conjugated linolenic acids can also be used as an animalfeed additive. The quality of meat grown for consumption is dependentupon many variables that ultimately influence market demand for theproduct. For instance, pork quality improvement is a primary focus ofthe pork industry. Quality variables include pork color, water holdingcapacity, size, chemical composition and firmness of lean and fattissue. Experiments have shown that the fat firmness of pork can beinfluenced by the addition of conjugated linoleic acid(18:2Δ^(9cis, 11trans) or Δ^(10trans, 12cis)) to swine diets (Eggert, J.M., et al. (1999) J. Anim. Sci. 77(Suppl):53; Thiel, R. C., et al.(1998)J. Anim. Sci. 76(Suppl):13; Wiegand, B. R., F. C. Parrrish Jr andJ. C. Sparks (1999) J. Anim. Sci. 77(Suppl):19; U.S. Pat. No. 5,554,646;and U.S. Pat. No. 5,851,572). Some experiments have also reportedimproved carcass leanness and the efficiency of feed utilization whenconjugated linoleic acid (CLA) is added as a supplement to the diet. Itis not known whether feeding of different conjugated fatty acids wouldhave similar effects. The present invention describes the production ofconjugated double bonds in 18:3 and 18:4 fatty acids which are derivedfrom 18:3 fatty acids in transgenic seeds that can be used as feedadditives.

Thus, the instant invention concerns animal feed comprising aningredient derived from the processing of any of the seeds obtainedplants or plant cells transformed with any of the chimeric genes. Theingredient or conjugated linolenic acid should be present in a carcassquality improving amount. A “carcass quality improving amount” is thatamount needed to improve the carcass quality of an animal. Theingredient can be a mixture of fatty acids obtained from such seeds.This mixture can be in any form suitable for use as a feed additive. Forexample, the mixture can be in the form of an oil whether or not it issaponified.

Also of interest is animal feed comprising oil obtained from any of theforegoing seeds. This invention also includes a method of improving thecarcass quality of an animal by supplementing a diet of the animal withany of the animal feeds discussed above.

In a further aspect the present invention also concerns an isolatednucleic acid fragment encoding a plant fatty acid modifying enzyme(DMFad2-1) associated with modification of the delta-12 position ofoleic acid to produce trans-linoleic acid, wherein said fragment or afunctionally equivalent subfragment thereof (a) hybridizes to any of thenucleotide sequences set forth in SEQ ID NO:10 under conditions ofmoderate stringency or (b) is at least 75% identical to a polypeptideencoded by any of the nucleotide sequences set forth in SEQ ID NO:11 ora functionally equivalent subfragment thereof as determined by acomparison method designed to detect homologous sequences.

This invention also concerns an isolated nucleic acid fragment encodinga plant fatty acid modifying enzyme wherein said enzyme modifies adelta-12 position of fatty acids and further wherein said fragment or afunctionally equivalent subfragment thereof (a) hybridizes to any of thenucleotide sequences set forth in SEQ ID NO:10 under conditions ofmoderate stringency or (b) is at least 75% identical to a polypeptideencoded by the nucleotide sequence set forth in SEQ ID NOs:11 or afunctionally equivalent subfragment thereof as determined by acomparison method designed to detect homologous sequences.

This invention also relates to the following:

a) an isolated nucleic acid fragment encoding a plant fatty acidmodifying enzyme associated with conjugated double bond formationcomprising the delta-12 position of fatty acids wherein said fragmentencodes a protein comprising any one of the amino acid sequences setforth in SEQ ID NO:11, as well as

b) an isolated nucleic acid fragment encoding a plant fatty acidmodifying enzyme wherein said enzyme modifies a delta-12 position offatty acids and further wherein said fragment or a functionallyequivalent subfragment thereof encodes a protein comprising any one ofthe amino acid sequences set forth in SEQ ID NO:11.

In another aspect, this invention concerns an isolated nucleic acidfragment encoding a plant fatty acid modifying enzyme associated withconjugated double bond formation comprising a the delta-12 position offatty acids or an isolated nucleic acid fragment encoding a plant fattyacid modifying enzyme wherein said enzyme modifies the delta-12 positionof the fatty wherein said fragments or a functionally equivalentsubfragments thereof hybridize to any of the isolated nucleic acidfragments or functionally equivalent subfragments thereof encoding aplant fatty acid modifying enzyme associated with conjugated double bondformation comprising the delta-12 position of fatty acids or associatedwith modification of the delta-12 position wherein said fragments orsubfragments encode a protein comprising any one of the amino acidsequences set forth in SEQ ID NO:11 and further wherein said fragmentsor subfragments (a) hybridize to these isolated nucleic acid fragmentsor functionally equivalent subfragments under conditions of moderatestringency or (b) is at least 75% identical to a polypeptide encoded byany of the foregoing isolated nucleic acid fragments or a functionallyequivalent subfragments thereof as determined by a comparison methoddesigned to detect homologous sequences. Examples of suitable comparisonmethods which detect homologous sequences are discussed above.

Also of interest is a chimeric gene comprising any of the instantisolated nucleic acid fragments, or functionally equivalent subfragmentsthereof, or a complement thereof operably linked to suitable regulatorysequences wherein expression of the chimeric gene results in productionof altered levels of the desired enzyme in a transformed host cell orplant.

The invention also relates to methods of using such isolated nucleicacid fragments, or functionally equivalent subfragments thereof, or thecomplement thereof, to alter the level of fatty acids comprising amodification of the delta-12 position of fatty acids in a host cell orplant which comprises:

(a) transforming a host cell or plant with any of the instant chimericgenes;

(b) growing the transformed host cell or plant under conditions suitablefor the expression of the chimeric gene; and

(c) selecting those transformed host cells or plants having alteredlevels of fatty acids comprising a modification at the delta-12position.

In still another aspect, this invention concerns a method for producingseed oil containing fatty acids comprising a modification at thedelta-12 position in the seeds of plants which comprises:

(a) transforming a plant cell with any of the instant chimeric genes;

(b) growing a fertile mature plant from the transformed plant cell ofstep (a);

(c) screening progeny seeds from the fertile plants of step (b) foraltered levels of fatty acids comprising a modification at the delta-12position; and

(d) processing the progeny seed of step (c) to obtain seed oilcontaining altered levels plant fatty acids comprising a modification atthe delta-12 position.

In still a further aspect, this invention concerns a method forproducing plant fatty acid modifying enzymes associated withmodification of the delta-12 position of fatty acids which comprises:

(a) transforming a microbial host cell with any of the instant chimericgenes;

(b) growing the transformed host cell under conditions suitable for theexpression of the chimeric gene; and

(c) selecting those transformed host cells containing altered levels ofprotein encoded by the chimeric gene.

The isolated nucleic acid fragments encoding fatty acid modifyingenzymes associated with conjugated double bond formation comprising thedelta-12 position of fatty acids in seeds of Dimorphotheca sinuata isprovided in SEQ ID NO:10, and the corresponding deduced amino acidsequences are provided in SEQ ID NO:11. Fatty acid modifying enzymesassociated with conjugated double bond formation comprising modificationof the delta-12 position of fatty acids from other plants fatty acidmodifying enzymes which are capable of modifying the delta-12 positionof a fatty acid can now be identified by when nucleotide sequencehybridizes to any of the nucleotide sequences set forth in SEQ ID NO:10under conditions of moderate stringency, as set forth above, or (b) isat least 75% identical to a polypeptide encoded by any of the nucleotidesequences set forth in SEQ ID NO:10 or a functionally equivalentsubfragment thereof as determined by a comparison method designed todetect homologous sequences.

Thus, the chimeric genes of the instant invention can be used to createtransgenic plants in which the fatty acid modifying enzymes which modifythe delta-12 position of fatty acids in seeds of Dimorphotheca sinuataare present at higher levels than normal or in cell types ordevelopmental stages in which it is not normally found. Also of interestare seeds obtained from such plants and oil obtained from these seeds.

Transgenic plants can be made in which fatty acid modifying enzymeassociated with modification of the delta-12 position of fatty acids ispresent at lower levels than normal or in cell types or developmentalstages in which it is not normally found. This would have the effect ofaltering the level of such fatty acids comprising a modified delta-12position in those cells. It may be desirable to reduce or eliminateexpression of a gene encoding such enzymes in plants for someapplications. In order to accomplish this, a chimeric gene designed forco-suppression of the endogenous enzyme can be constructed by linking agene or gene fragment encoding a fatty acid modifying enzyme associatedwith modification of the delta-12 position of fatty acids to plantpromoter sequences. Alternatively, a chimeric gene designed to expressantisense RNA for all or part of the instant nucleic acid fragment, canbe constructed by linking the gene or a gene fragment in reverseorientation to plant promoter sequences. Either the co-suppression orantisense chimeric genes could be introduced into plants viatransformation wherein expression of the corresponding endogenous genesare reduced or eliminated.

When overexpressed in plant cells, the fatty acid modifying enzymesassociated with modification of the delta-12 position of fatty acids inseeds of Dimorphotheca sinuata can be useful for causing thebiosynthesis and accumulation of fatty acids with conjugated doublebonds, such as calendic acid, in those cells. It is particularly usefulto use fatty acid modifying enzymes associated with modification of thedelta-12 position of fatty acids in seeds of Dimorphotheca sinuata toproduce fatty acids containing conjugated double bonds in the cells ofthe seeds of oilseed crop plants. The modification of the delta-12position by DMFad2-1 leads to an intermediate (trans-linoleic acid) thatis the precursor to dimorphecolic acid.

Overexpression of fatty acid modifying enzymes associated withmodification of the delta-12 position of fatty acids in seeds ofDimorphotheca sinuata may be accomplished by first constructing achimeric gene in which the coding region of cDNAs for fatty acidmodifying enzymes associated with modification of the delta-12 positionof fatty acids in seeds of Dimorphotheca sinuata is operably linked to apromoter capable of directing expression of a gene in the desiredtissues at the desired stage of development. For reasons of convenience,the chimeric gene may comprise a promoter sequence and translationleader sequence derived from the same gene. 3′ non-coding sequencesencoding transcription termination signals must also be provided. Theinstant chimeric genes may also comprise one or more introns in order tofacilitate gene expression.

Vectors, such as plasmid vectors, comprising the instant chimeric genescan then be constructed. The choice of plasmid vector is dependent uponthe method that will be used to transform host plants. The skilledartisan is well aware of the genetic elements that must be present onthe plasmid vector in order to successfully transform, select andpropagate host cells or plants containing the chimeric gene. The skilledartisan will also recognize that different independent transformationevents will result in different levels and patterns of expression (Joneset al., (1985) EMBO J. 4:2411-2418; De Almeida et al., (1989) Mol. Gen.Genetics 218:78-86), and thus that multiple events must be screened inorder to obtain lines displaying the desired expression level andpattern. Such screening may be accomplished by Southern analysis of DNA,Northern analysis of mRNA expression, Western analysis of proteinexpression, or phenotypic analysis.

For some applications it may be useful to direct the instant fatty acidmodifying enzymes associated with modification of the delta-12 positionof fatty acids in seeds of Dimorphotheca sinuata to different cellularcompartments, or to facilitate its secretion from the cell. It is thusenvisioned that the chimeric genes described above may be furthersupplemented by altering the coding sequences to encode fatty acidmodifying enzymes associated with modification of the delta-12 positionof fatty acids in seeds of Dimorphotheca sinuata disclosed herein withappropriate intracellular targeting sequences, such as transit sequences(Keegstra, K. (1989) Cell 56:247-253), signal sequences or sequencesencoding endoplasmic reticulum localization (Chrispeels, J. J., (1991)Ann. Rev. Plant Phys. Plant Mol. Biol. 42:21-53), or nuclearlocalization signals (Raikhel, N. (1992) Plant Phys. 100:1627-1632)added and/or with targeting sequences that are already present removed.While the references cited give examples of each of these, the list isnot exhaustive and more targeting signals of utility may be discoveredin the future.

The nucleic acid fragments of the instant invention, or functionallyequivalent subfragment thereof, may be used to isolate cDNAs and othernucleic acid fragments encoding homologous fatty acid modifying enzymesfrom the same or other plant species. Isolation of homologous genesusing sequence-dependent protocols is well known in the art. Examples ofsequence-dependent protocols include, but are not limited to, methods ofnucleic acid hybridization, and methods of DNA and RNA amplification asexemplified by various uses of nucleic acid amplification technologies(e.g., polymerase chain reaction, ligase chain reaction). The term“conserved sequence(s)” as used herein encompasses both strictconservation as well as conservation of a majority of the sequences usedin an alignment, for example, conservation with respect to a consensussequence.

Thus, in still a further aspect this invention concerns a method toisolate nucleic acid fragments and functionally equivalent subfragmentsthereof encoding a plant fatty acid modifying enzyme associated withmodification of the delta-12 position of fatty acids comprising:

(a) comparing SEQ ID NO:11 and other plant fatty acid modifying enzyme.polypeptide sequences;

(b) identifying conserved sequences of 4 or more amino acids obtained instep (a);

(c) designing degenerate oligomers based on the conserved sequencesidentified in step (b); and

(d) using the degenerate oligomers of step (s) to isolate sequencesencoding a plant fatty acid modifying enzyme or a portion thereofassociated with modification of the delta-12 position of fatty acids bysequence dependent protocols.

For example, genes encoding homologous fatty acid modifying enzymes,either as cDNAs or genomic DNAs, could be isolated directly by using allor a portion of the instant nucleic acid fragments as DNA hybridizationprobes to screen libraries from any desired plant employing methodologywell known to those skilled in the art. Specific oligonucleotide probesbased upon the instant nucleic acid sequences can be designed andsynthesized by methods known in the art (Sambrook). Moreover, the entiresequences can be used directly to synthesize DNA probes by methods knownto the skilled artisan such as random primers DNA labeling, nicktranslation, or end-labeling techniques, or RNA probes using availablein vitro transcription systems. In addition, specific primers can bedesigned and used to amplify a part of or full-length of the instantsequences. The resulting amplification products can be labeled directlyduring amplification reactions or labeled after amplification reactions,and used as probes to isolate full length cDNA or genomic fragmentsunder conditions of appropriate stringency.

In addition, two short segments of the instant nucleic acid fragmentsmay be used in polymerase chain reaction protocols to amplify longernucleic acid fragments encoding homologous genes from DNA or RNA. Thepolymerase chain reaction may also be performed on a library of clonednucleic acid fragments wherein the sequence of one primer is derivedfrom the instant nucleic acid fragments, and the sequence of the otherprimer takes advantage of the presence of the polyadenylic acid tractsto the 3′ end of the mRNA precursor encoding plant genes. Alternatively,the second primer sequence may be based upon sequences derived from thecloning vector. For example, the skilled artisan can follow the RACEprotocol (Frohman et al., (1988) PNAS USA 85:8998) to generate cDNAs byusing PCR to amplify copies of the region between a single point in thetranscript and the: 3′ or 5′ end. Primers oriented in the 3′ and 5′directions can be designed from the instant sequences. Usingcommercially available 3′ RACE or 5′ RACE systems (BRL), specific 3′ or5′ cDNA fragments can be isolated (Ohara et al., (1989) PNAS USA86:5673; Loh et al., (1989) Science 243:217). Products generated by the3′ and 5′ RACE procedures can be combined to generate full-length cDNAs(Frohman, M. A. and Martin, G. R., (1989) Techniques 1:165).

Thus, other nucleic acid fragments encoding enzymes associated withmodification of the delta-12 position of fatty acids can be identifiedusing any of the general methodologies described above. For example, ageneral group of fatty acid desaturase (FAD) related cDNAs, can beidentified and a specific subset of those cDNAs encoding enzymesinvolved in modification of the delta-12 position of fatty acids can bedetected or screened by transformation. A group of cDNA sequencesencoding fatty acid desaturase-like enzymes can be identified usinglow-stringency hybridization (for example 2×SSC, 0.1% SDS, 60° C.) witha probe corresponding to any known FAD sequence, and/or all-or-part ofthe sequences presented in any of SEQ ID NO:10. Alternatively, randomlysequenced cDNAs can be analyzed by a computer program designed to detecthomologous sequences, such as, but not limited to, BLAST or gapped BLAST(using standard default parameters). BLAST (Basic Local Alignment SearchTool; Altschul et al. (1993) J. Mol. Biol. 215:403-410) searches forsimilarity to sequences contained in the BLAST “nr” database (comprisingall non-redundant GenBank CDS translations, sequences derived from the3-dimensional structure Brookhaven Protein Data Bank, the last majorrelease of the SWISS-PROT protein sequence database, EMBL, and DDBJdatabases). Test sequences are analyzed for similarity to all publiclyavailable DNA sequences contained in the “nr” database using the BLASTNalgorithm provided by the National Center for Biotechnology Information(NCBI). The DNA sequences are translated in all reading frames andcompared for similarity to all publicly available protein sequencescontained in the “nr” database using the BLASTX algorithm (Gish andStates (1993) Nature Genetics 3:266-272) provided by the NCBI. Forconvenience, the P-value (probability), or “pLog” (the negative of thelogarithm of the P-value), is given as a measure of similarity betweenthe two sequences. A test sequence and a sequence contained in thesearched databases are compared, and the probability that the twosequences are related only by chance is calculated by BLAST and reportedas a “pLog” value. Accordingly, the greater the pLog value, the greaterthe likelihood that the cDNA sequence and the BLAST “hit” representhomologous proteins. Sequences with pLogs greater than 5, or preferablygreater than 10, or more preferably greater than 15, and most preferablygreater than 20, that are defined as FADs or lipid desaturases arecandidates. cDNAs encoding enzymes associated with modification of thedelta-12 position of fatty acids can be identified from the candidatepools using transformation screening. Individual cDNAs are inserted intoexpression vectors and transformed into yeast or plant host cells usingmethods well known to those skilled in the art (see Examples 3, 4, 6, 7,and 8). Production of fatty acids containing conjugated double bonds isconfirmed by GC-MS analyses as described in the Examples 3 and 4. Yeastor plant tissue culture cells are preferred for initial screening due tospeed and the ease with which they can be handled when dealing withlarge numbers of transformants and the appropriate cell biology andeukaryotic cell physiology.

The instant fatty acid modifying enzymes associated with modification ofthe delta-12 position of fatty acids in seeds of Dimorphotheca sinuataproduced in heterologous host cells or plants, particularly in the cellsof microbial hosts, can be used to prepare antibodies to the fatty acidmodifying enzymes associated with modification of the delta-12 positionof fatty acids in seeds of Dimorphotheca sinuata by methods well knownto those skilled in the art. The antibodies are useful for detecting theinstant fatty acid modifying enzymes associated with modification of thedelta-12 position of fatty acids in seeds of Dimorphotheca sinuata insitu in cells or in vitro in cell extracts. Preferred heterologous hostcells for production of the instant fatty acid modifying enzymesassociated with modification of the delta-12 position of fatty acids inseeds of Dimorphotheca sinuata are microbial hosts. Microbial expressionsystems and expression vectors containing regulatory sequences thatdirect high level expression of foreign proteins are well known to thoseskilled in the art. Any of these could be used to construct chimericgenes for production of the instant fatty acid modifying enzymesassociated with modification of the delta-12 position of fatty acids inseeds of Dimorphotheca sinuata. These chimeric genes could then beintroduced into appropriate microorganisms via transformation to providehigh level expression of the encoded fatty acid modifying enzymesassociated with modification of the delta-12 position of a fatty acid inseeds Dimorphotheca sinuata. An example of a vector for high levelexpression of the instant fatty acid modifying enzymes associated withmodification of the delta-12 position of fatty acids in seeds ofDimorphotheca sinuata in a bacterial host is discussed below in Example8.

EXAMPLES

The present invention is further defined in the following Examples, inwhich all parts and percentages are by weight and degrees are Celsius,unless otherwise stated. It should be understood that these Examples,while indicating preferred embodiments of the invention, are given byway of illustration only. From the above discussion and these Examples,one skilled in the art can ascertain the essential characteristics ofthis invention, and without departing from the spirit and scope thereof,can make various changes and modifications of the invention to adapt itto various usages and conditions.

Example 1 Composition of cDNA Libraries; Isolation and Sequencing ofcDNA Clones

cDNA libraries representing mRNAs from developing seeds of Calendulaofficinalis were prepared. The seeds chosen were actively accumulatingfatty acids with conjugated double bonds. The libraries were preparedusing a Uni-ZAP™ XR kit according to the manufacturer's protocol(Stratagene Cloning Systems, La Jolla, Calif.), except that cDNAs werecloned into the EcoRI and XhoI sites of the bacterial vector pBluescriptSK(−) rather than into a phage vector. Libraries were maintained in E.coli DH10B cells (Life Technologies, Gaithersburg, Md.). cDNA insertsfrom randomly picked bacterial colonies containing recombinantpBluescript plasmids were grown up and plasmid purified. cDNAs weresequenced using primers specific for vector sequences flanking theinserted cDNA sequences. Insert DNAs were sequenced in dye-primersequencing reactions to generate partial cDNA sequences (expressedsequence tags or “ESTs”; see Adams, M. D. et al., (1991) Science252:1651) using a Perkin Elmer Model 377 fluorescent sequencer. Theresulting ESTs were analyzed using computational methods as describedbelow.

Example 2 Identification and Characterization of cDNA Clones

ESTs encoding Calendula officinalis fatty acid modifying enzymes wereidentified by conducting BLAST (Basic Local Alignment Search Tool;Altschul, S. F., et al., (1993) J. Mol. Biol. 215:403-410) searches forsimilarity to sequences contained in the BLAST “nr” database (comprisingall non-redundant GenBank coding sequence [“CDS”] translations,sequences derived from the 3-dimensional structure Brookhaven ProteinData Bank, the last major release of the SWISS-PROT protein sequencedatabase, EMBL, and DDBJ databases). The cDNA sequences obtained inExample 1 were analyzed for similarity to all publicly available DNAsequences contained in the “nr” database using the BLASTN algorithmprovided by the National Center for Biotechnology Information (NCBI).The DNA sequences were translated in all reading frames and compared forsimilarity to all publicly available protein sequences contained in the“nr” database using the BLASTX algorithm (Gish, W. and States, D. J.(1993) Nature Genetics 3:266-272) provided by the NCBI. For convenience,the P-value (probability) of observing a match of a cDNA sequence to asequence contained in the searched databases merely by chance ascalculated by BLAST are reported herein as “pLog” values, whichrepresent the negative of the logarithm of the reported P-value.Accordingly, the greater the pLog value, the greater the likelihood thatthe cDNA sequence and the BLAST “hit” represent homologous proteins.

The BLASTX search using sequence information derived from the entireCalendula officinalis clone ecs1c.pk009.n14 (CalFad2-1) revealed strongsimilarity to the proteins encoded by cDNAs for omega-6 fatty aciddesaturases from Petroselinum crispum (Genbank Accession No. gi2501790;pLog=133.00) and Brassica juncea (Genbank Accession No. gi3334184;pLog=127.00). The BLASTX search using sequence information derived fromthe entire Calendula officinalis clone ecs1c.pk008.a24 (CalFad2-2)revealed strong similarity to the proteins encoded by cDNAs for delta-12fatty acid desaturases from Borago officinalis (Genbank Accession No.gi3417601; pLog=135.00) and Brassica carinata (Genbank Accession No.gi4378875; pLog=135.00). SEQ ID NO:1 shows the nucleotide sequence ofthe entire Calendula officinalis cDNA in clone ecs1c.pk009.n14; thededuced amino acid sequence is shown in SEQ ID NO:2. SEQ ID NO:3 showsthe nucleotide sequence of the entire Calendula officinalis cDNA inclone ecs1c.pk008.a24; the deduced amino acid sequence is shown in SEQID NO:4. Sequence alignments and BLAST scores and probabilities indicatethat the instant nucleic acid fragments encode Calendula officinalisproteins that is structurally related to the omega-6 and delta-12 classof fatty acid desaturases. The clones for these proteins were designatedCalFad2-1 and CalFad2-2, respectively.

Example 3 Expression of CalFad2-1, a Diverged Calendula Fad2, in TobaccoCells

To characterize the activity of the CalFad2-1 in transgenic plant cells,the cDNA (ecs1c.pk009.n14) encoding this enzyme was expressed in tobaccocallus with the gene under control of the cauliflower mosaic virus 35Spromoter. The open-reading frame of the cDNA for CalFad2-1 was amplifiedby PCR to generate flanking 5′ BamHI and 3′ SstI restriction enzymesites for cloning into the plant expression vector. The sequence of thesense oligonucleotide used in the amplification reaction was5′-tttgagctcTACACCTAGCTACGTACCATG-3′ (SEQ ID NO:16), and the sequence ofthe antisense oligonucleotide was 5′-tttggatccTCACGGTACTGATGATGGCAC -3′(SEQ ID NO:17) [Note: the bases in lower case contain the addedrestriction sites, which are underlined, and flanking sequence tofacilitate restriction enzyme digestion]. The design of the PCR primerswas based on the sequence of the CalFad2-1 cDNA shown in SEQ ID NO:1.Thirty cycles of PCR amplification were conducted in a 100 μl volumeusing Pfu polymerase (Stratagene) and 25 ng of pBluescript SK(−)containing the CalFad2-1 cDNA. The product from this reaction wassubcloned into pPCR-Script AMP (Stratagene). Following restrictiondigestion with BamHI and SstI, the PCR product was moved frompPCR-Script AMP into the corresponding sites of the plant expressionvector pBI121 (Clontech). The vector pBI121 is used for constitutiveexpression of transgenes mediated by the cauliflower mosaic virus 35Spromoter. This vector contains right and left border regions flankingthe inserted gene fusion to facilitate stable Agrobacterium-mediatedtransformation of the host plant cell and also contains within theborder regions a nopaline phosphotransferase II (NPTII) gene undercontrol of the cauliflower mosaic virus 35S promoter to provide forselection of transformed plant cells by kanamycin resistance. Theresulting construct containing the 35S promoter fused with CalFad2-1cDNA was transformed into Agrobacterium tumefaciens LBA4404 cells.Cultures derived from these cells were used for transformation oftobacco (Nicotiana tabacum cv. Xanthi) leave disks according to theprotocol described by Rogers, S. G., Horsch, R. B., and Fraley, R. T.(1986) Methods Enzymol. 118: 627-648.

Kanamycin-resistant tobacco callus that resulted from the transformationwas examined for the presence of calendic acid arising from the activityof CalFad2-1. Fatty acid methyl esters were prepared by homogenizationof the transgenic tobacco callus in 1% (w/v) sodium methoxide inmethanol using methods described by Hitz et al. (1994) Plant Physiol.105:635-641. The recovered fatty acid methyl esters were then analyzedusing a Hewlett-Packard 6890 chromatograph fitted with an Omegawax 320column (30 m×0.32 mm inner diameter; Supelco). The oven temperature wasprogrammed from 220° C. (2 min hold) to 240° C. at a rate of 20° C./min.The retention time of methyl calendic acid in extracts of tobacco calluswas compared with that of methyl calendic acid in seeds of Calendulaofficinalis. Gas chromatography-mass spectrometry (GC-MS) was alsoperformed to confirm the identity of calendic acid in tobacco callusexpressing CalFad2-1. Fatty acid methyl prepared from the transgenictobacco callus was analyzed with an HP6890 interfaced with a HP5973(Hewlett-Packard) mass selective detector. Compounds were resolved usingHP-5 column (30 m×0.25 mm inner diameter) with the oven temperatureprogrammed from 185° C. (2-min hold) to 215° C. at a rate of 5° C./min.The mass spectrum of methyl calendic acid from Calendula seed extractsis characterized by an abundant molecular ion of 292 m/z.

In fatty acid methyl esters prepared from the stably transformed tobaccocallus, methyl calendic acid was detected in amounts of up to 11.4% ofthe total fatty acids (FIG. 3). The peak identified as methyl calendicacid in callus expressing CalFad2-1 had a retention time and massspectrum that was identical to those of methyl calendic acid inCalendula officinalis seeds. No methyl calendic acid was detected intobacco callus transformed with the vector lacking cDNA insert. Theseresults further demonstrate the ability to produce calendic acid bytransgenic expression of CalFad2-1.

Example 4 Expression of Calendula officinalis clones CalFad2-1 andCalFad2-2 in Saccharomyces cerevisiae

The Calendula officinalis clones CalFad2-1 and CalFad2-2 were digestedwith the restriction enzymes EcoRI and XhoI. The resulting DNA fragmentscontaining the entire cDNA inserts were purified by agarose gelelectrophoresis. The purified cDNAs were ligated into the EcoRI and XhoIsites of the Saccharomyces cerevisiae expression vector pYES2(Invitrogen) using T4 DNA ligase (New England Biolabs). The resultingplasmids pYes2/CalFad2-1 and pYes2/CalFad2-2 were introduced intoSaccharomyces cerevisiae INVSc1 (Invitrogen Corp.) cells by lithiumacetate-mediated transformation [Sherman F, Fink G R, Hicks J B, Methodsin Yeast Genetics: A Laboratory Course Manual, Cold Spring Harbor Lab.Press, Plainview, N.Y. (1987)]. Transformed cells were selected fortheir ability to grow in the absence of uracil. Individual colonies oftransformed cells were then grown for 2 days at 30° C. in growth medialacking uracil [0.17% (w/v) yeast nitrogen base without amino acids(Difco), 0.5% (w/v) ammonium sulfate, and 0.18% SC-URA (Bio101)]supplemented with glycerol and glucose to a final concentration of 5%(v/v) and 0.5% (w/v), respectively. Cells were then washed twice in thegrowth media described above that was supplemented instead withgalactose to a final concentration of 2% (w/v). The washed cells werethen diluted to O.D.₆₀₀˜0.2 in the galactose-containing growth mediathat also contained Tergitol NP-40 (Sigma) at a concentration of 0.2%(w/v). Aliquots of these cells were grown without exogenous fatty acidsor with the addition of linoleic acid (18:2Δ^(9cis, 12cis)) to a finalconcentration of 2 mM. Following 4 days of growth at 16° C., the S.cerevisiae cells were harvested and examined for the accumulation offatty acids containing conjugated double bonds as described in Example4. In cells grown in media containing linoleic acid, calendic acid(18:3Δ^(8tran, 10trans, 12cis)) was detected in amounts of up to 2.9%(w/w) of the total fatty acids of cultures expressing CalFad2-1 (FIG. 3)and in amounts of up to 0.2% of the total fatty acids of culturesexpressing CalFad2-2. The identity of calendic acid was established bycomparison of the gas chromatographic retention time and mass spectrumof its methyl ester derivative with that of methyl calendic acid inextracts of Calendula seeds. No calendic acid was detected in culturesharboring the expression vector without a cDNA insert or in cells grownin the absence of exogenous linoleic acid. These data are consistentwith linoleic acid serving as the substrate for calendic acid synthesisvia the activity of the Calendula officinalis fatty acid modifyingenzyme associated with conjugated double bond formation and modificationof the delta-9 position of fatty acids. In this reaction, the delta-9double bond of linoleic acid is converted by the activity of CalFad2-1or CalFad2-2 to delta-8 and delta-10 double bonds. The resulting fattyacid, calendic acid, contains delta-8, delta-10, and delta-12 doublebonds in conjugation. CalFad2-1 and CalFad2-2 are thus distinct from allpreviously reported Fad2-related polypeptides by their ability to modifythe delta-9 rather than the delta-12 position of a fatty acid.

Example 5 Comparison of the Proteins from Calendula with Impatiens,Momordica, and Chrysobalanus Enzymes Involved in Conjugated Fatty acidBond Formation, as well as Members of the Omega-6 Desaturase Class ofEnzymes

The deduced amino acid sequences from cDNA clones CalFad2-1, CalFad2-2,ImpFad2 H8, and MomFad2 were compared to the deduced amino acidsequences encoding (i) a known fatty acid desaturase from soybean (WorldPatent Publication No. WO94/11516) and (ii) a fatty acid hydroxylasefrom castor bean (van de Loo, F. J. et al. (1995) Proc. Natl. Acad. Sci.U.S.A. 92 (15):6743-6747) using the multiple sequence comparison programMegalign (v3.1.7) from the Lasergene□ software package (DNASTAR Inc.,Madison, WI) and the Clustal method of alignment (default programparameters). The aligned sequences are shown in FIGS. 2A-2D. All sevensequences, including those of the proteins from Calendula officinalisare related by eight highly conserved histidine residues that arebelieved to be part of the binding site for the two-iron cluster that isrequired in the active site of this class of enzymes (Shanklin, J. etal. (1994) Biochemistry 33:12787-12793). These conserved residues areidentified as boxed elements in FIGS. 2A-2D. The amino acid sequenceencoded by the Impatiens balsamina cDNA clone ImpH8Fad2 is 57.0%identical to the soybean sequence and 55.2% identical to the castorsequence. The amino acid sequence encoded by the Momordica charantiacDNA clone MomFad2 is 56.7% identical to the soybean sequence and 53.5%identical to the castor sequence. Overall, the sequence similarityshared by the two Calendula officinalis proteins is 94.6%. CalFad2-1 is45.5% identical to the soybean sequence and 44.1% identical to thecastor sequence. CalFad2-2 is 46.8% identical to the soybean sequenceand 44.1% identical to the castor sequence.

The residue immediately adjacent to the first histidine box in bothCalendula enzymes is a glycine (as indicated by an asterisk in FIGS.2A-2D). A glycine in this position is only observed in ω⁶-oleic aciddesaturase-related enzymes that have diverged functionality, such as thecastor oleic acid hydroxylase (van de Loo, F. J. et al. (1995) Proc.Natl. Acad. Sci. U.S.A. 92:6743-6747) and the Crepis palaestinaepoxidase (Lee, M. et al. (1998) Science 280:915-918). Given thisfeature of its primary structure and its more distant relation to knownω⁶-oleic acid desaturases, it is believed that the polypeptides encodedby CalFad2-1 and CalFad2-2 are associated with conjugated double bondformation and are not conventional fatty acid desaturases (like thesoybean sequence in FIGS. 2A-2D). The Impatiens enzyme (ImpFad2H8) isknown to make eleostearic acid, a conjugated fatty acid, and alsocontains this glycine for alanine substitution.

Thus, changes in a comparatively small number of amino acid residues inconserved regions of the protein are sufficient to alter the activity inthis class of enzymes from one of introducing a double bond (i.e., adesaturase) to one of introducing an hydroxyl group (i.e., ahydroxylase) or to one that is active in converting polyunsaturatedfatty acids to fatty acids containing multiple conjugated double bonds.

Example 6 Expression of Chimeric Genes in Monocot Cells

The oil storing tissues of most grass seeds are the embryo and itsattending tissues the scutellum and to some extent the aleurone.Promoter sequences such as those controlling expression of the storageproteins Globulin 1 (Belanger, S. C. and Kriz, A. L (1989) PlantPhysiol. 91:636-643) and Globulin 2 (Wallace, N. H. and Kriz, A. L.(1991) Plant Physiol. 95:973-975) are appropriate for the expression ofchimeric genes in these tissues.

A chimeric gene comprising a cDNA encoding fatty acid modifying enzymesassociated with conjugated double bond synthesis comprising the delta-9position in seeds of Calendula officinalis in sense orientation withrespect to the maize Globulin 2 promoter that is located 5′ to the cDNAfragment, and the Globulin 2, 3′ end that is located 3′ to the cDNAfragment, can be constructed. The cDNA fragment of this gene may begenerated by polymerase chain reaction (PCR) of the cDNA clone usingappropriate oligonucleotide primers. Cloning sites can be incorporatedinto the oligonucleotides to provide proper orientation of the DNAfragment when inserted into the correctly designed expression vector.

Such expression vectors should include genetic sequences elementsconferring an origin of replication for the plasmid in its host, a genecapable of conferring a selectable trait such as autotrophy orantibiotic tolerance to the host cell carrying the plasmid and thepromoter sequences for expression of desired genes in host plant cells.Further design features may include unique restriction endonucleaserecognition sites between the elements of the plant gene promoterelements to allow convenient introduction genes to be controlled bythose elements. Plants that can serve as suitable hosts include, but arenot limited to, corn, rice, wheat, and palm.

The chimeric genes constructed as above can then be introduced into corncells by the following procedure. Immature corn embryos can be dissectedfrom developing caryopses derived from crosses of the inbred corn linesH99 and LH132. The embryos are isolated 10 to 11 days after pollinationwhen they are 1.0 to 1.5 mm long. The embryos are then placed with theaxis-side facing down and in contact with agarose-solidified N6 medium(Chu et al., (1975) Sci. Sin. Peking 18:659-668). The embryos are keptin the dark at 27°. Friable embryogenic callus consisting ofundifferentiated masses of cells with somatic proembryoids and embryoidsborne on suspensor structures proliferates from the scutellum of theseimmature embryos. The embryogenic callus isolated from the primaryexplant can be cultured on N6 medium and sub-cultured on this mediumevery 2 to 3 weeks.

The plasmid, p35S/Ac (obtained from Dr. Peter Eckes, Hoechst Ag,Frankfurt, Germany) may be used in transformation experiments in orderto provide for a selectable marker. This plasmid contains the Pat gene(see European Patent Publication 0 242 236) which encodesphosphinothricin acetyl transferase (PAT). The enzyme PAT confersresistance to herbicidal glutamine synthetase inhibitors such asphosphinothricin. The pat gene in p35S/Ac is under the control of the35S promoter from Cauliflower Mosaic Virus (Odell et al. (1985) Nature313:810-812) and the 3′ region of the nopaline synthase gene from theT-DNA of the Ti plasmid of Agrobacterium tumefaciens.

The particle bombardment method (Klein et al., (1987) Nature 327:70-73)may be used to transfer genes to the callus culture cells. According tothis method, gold particles (1 μm in diameter) are coated with DNA usingthe following technique. Ten μg of plasmid DNAs are added to 50 μL of asuspension of gold particles (60 mg per mL). Calcium chloride (50 μL ofa 2.5 M solution) and spermidine free base (20 μL of a 1.0 M solution)are added to the particles. The suspension is vortexed during theaddition of these solutions. After 10 minutes, the tubes are brieflycentrifuged (5 sec at 15,000 rpm) and the supernatant removed. Theparticles are resuspended in 200 μL of absolute ethanol, centrifugedagain and the supernatant removed. The ethanol rinse is performed againand the particles resuspended in a final volume of 30 μL of ethanol. Analiquot (5 μL) of the DNA-coated gold particles can be placed in thecenter of a Kapton® flying disc (Bio-Rad Labs). The particles are thenaccelerated into the corn tissue with a Biolistic® PDS-1000/He (Bio-RadInstruments, Hercules Calif.), using a helium pressure of 1000 psi, agap distance of 0.5 cm and a flying distance of 1.0 cm.

For bombardment, the embryogenic tissue is placed on filter paper overagarose-solidified N6 medium. The tissue is arranged as a thin lawn andcovered a circular area of about 5 cm in diameter. The petri dishcontaining the tissue can be placed in the chamber of the PDS-1000/Heapproximately 8 cm from the stopping screen. The air in the chamber isthen evacuated to a vacuum of 28 inches of Hg. The macrocarrier isaccelerated with a helium shock wave using a rupture membrane thatbursts when the He pressure in the shock tube reaches 1000 psi.

Seven days after bombardment the tissue can be transferred to N6 mediumthat contains gluphosinate (2 mg per liter) and lacks casein or proline.The tissue continues to grow slowly on this medium. After an additional2 weeks the tissue can be transferred to fresh N6 medium containinggluphosinate. After 6 weeks, areas of about 1 cm in diameter of activelygrowing callus can be identified on some of the plates containing theglufosinate-supplemented medium. Calli may continue to grow whensub-cultured on the selective medium.

Plants can be regenerated from the transgenic callus by firsttransferring clusters of tissue to N6 medium supplemented with 0.2 mgper liter of 2,4-D. After two weeks the tissue can be transferred toregeneration medium (Fromm et al., (1990) Bio/Technology 8:833-839).

Example 7 Expression of Chimeric Genes in Dicot Cells

Fatty acid modifying enzymes associated with conjugated double bondsynthesis comprising the delta-9 position in seeds of Calendulaofficinalis can be expressed in cells of dicots that normally producestorage lipid by the construction of appropriate chimeric genes followedby stable introduction of those genes into the host plant. An example ofthis method is the seed specific expression in soybean of fatty acidmodifying enzymes associated with conjugated double bond synthesis inseeds of Calendula officinalis. Other plants that can be used include,but are not limited to, oilseed Brassica species, peanut, sunflower,safflower, cotton, flax, and cocoa.

A plasmid pKS 18HH containing chimeric genes to allow expression ofHygromycin B Phosphotransferase in certain bacteria and in plant cellscan be constructed from the following genetic elements: a) T7Promoter+Shine-Delgarno/Hygromycin B Phosphotransferase (HPT)/T7Terminator Sequence, b) 35S Promoter from cauliflower mosaic virus(CaMV)/Hygromycin B Phosphotransferase (HPT)/Nopaline Synthase (NOS3′from Agrobacterium tumefaciens T-DNA, and c) pSP72 plasmid vector [fromPromega] with β-lactamase coding region (ampicillin resistance gene)removed.

The Hygromycin B Phosphotransferase gene can be amplified by PCR from E.coli strain W677, which contains a Klebsiella derived plasmid pJR225.Starting with the pSP72 vector the elements are assembled into a singleplasmid using standard cloning methods (Maniatis).

Plasmid pKS 18HH thus contains the T7 promoter/HPT/T7 terminatorcassette for expression of the HPT enzyme in certain strains of E. coli,such as NovaBlue(DE3) [from Novagen], that are lysogenic for lambda DE3(which carries the T7 RNA Polymerase gene under lacV5 control). PlasmidpKS18HH also contains the 35S/HPT/NOS cassette for constitutiveexpression of the HPT enzyme in plants, such as soybean. These twoexpression systems allow selection for growth in the presence ofhygromycin to be used as a means of identifying cells that contain theplasmid in both bacterial and plant systems.

pKS18HH also contains three unique restriction endonuclease sitessuitable for the cloning of other chimeric genes into this vector.

A plasmid for expression of the cDNA encoding fatty acid modifyingenzymes associated with conjugated double bond synthesis in seeds ofCalendula officinalis is made to be under the control of a soybeanβ-conglycinin promoter (Beachy et al., (1985) EMBO J. 4:3047-3053). Theconstruction of this vector is facilitated by the use of plasmids pCW109and pML18, both of which have been described (see World PatentPublication No. WO94/11516).

A unique Not I site is introduced into the cloning region between theβ-conglycinin promoter and the phaseolin 3′ end in pCW109 by digestionwith Nco I and Xba I followed by removal of the single stranded DNA endswith mung bean exonuclease. Not I linkers (New England Biochemicalcatalog number NEB 1125) are ligated into the linearized plasmid toproduce plasmid pAW35. The single Not I site in pML18 is destroyed bydigestion with Not I, filling in the single stranded ends with dNTP'sand Klenow fragment followed by re-ligation of the linearized plasmid.The modified pML18 is then digested with Hind III and treated withCalFad intestinal phosphatase.

The β-conglycinin:Not I:phaseolin expression cassette in pAW35 isremoved by digestion with Hind III and the 1.79 kB fragment is isolatedby agarose gel electrophoresis. The isolated fragment is ligated intothe modified and linearized pML18 construction described above. A clonewith the desired orientation was identified by digestion with Not I andXba I to release a 1.08 kB fragment indicating that the orientation ofthe β-conglycin transcription unit is the same as the selectable markertranscription unit. The resulting plasmid is given the name pBS19.

Hind III is one of the unique cloning sites available in pKS18HH. Toassemble the final expression cassette pBS19 and pKS18HH are bothdigested with Hind III. The β-conglycinin containing fragment from pBS19is isolated by gel electrophoresis and ligated into the digested pKS18HHwhich had been treated with CalFad alkaline phosphatase. The resultingplasmid is named pRB20.

The PCR products amplified from clones for the Calendula polypeptides(described in Example 3 above) are digested with restriction enzymes tocleave the sites designed into the PCR primers. Plasmid pRB20 is alsodigested in a manner compatible with conventional cloning sites for theintroduction of the PCR fragments. After phosphatase treatment of thelinearized pRB20, PCR products are ligated into pRB20 and the ligationmixtures are used to transform E. coli strain DH10B. Colonies areselected and grown in liquid media for preparation of plasmid DNA.Digestion of the plasmid DNAs with an enzyme diagnostic for correctorientation of the coding sequences relative to the β-conglycininpromoter identifies clones for use in soybean transformation.

Soybean embryos are then transformed with the expression vectorcomprising sequences encoding Calendula polypeptides described above. Toinduce somatic embryos, cotyledons, 3-5 mm in length dissected fromsurface sterilized, immature seeds of a soybean cultivar, such as A2872,can be cultured in the light or dark at 26° on an appropriate agarmedium for 6-10 weeks. Somatic embryos which produce secondary embryosare then excised and placed into a suitable liquid medium. Afterrepeated selection for clusters of somatic embryos that multiplied asearly, globular staged embryos, the suspensions are maintained asdescribed below.

Soybean embryogenic suspension cultures are maintained in 35 mL liquidmedia on a rotary shaker, 150 rpm, at 26° with fluorescent lights on a16:8 hour day/night schedule. Cultures are subcultured every two weeksby inoculating approximately 35 mg of tissue into 35 mL of liquidmedium.

Soybean embryogenic suspension cultures may then be transformed by themethod of particle gun bombardment (Kline et al. (1987) Nature (London)327:70, U.S. Pat. No. 4,945,050). A Du Pont Biolistic™ PDS 1000/HEinstrument (helium retrofit) can be used for these transformations.

A selectable marker gene which can be used to facilitate soybeantransformation is a chimeric gene composed of the 35S promoter fromCauliflower Mosaic Virus (Odell et al.(1985) Nature 313:810-812), thehygromycin phosphotransferase gene from plasmid pJR225 (from E. coli;Gritz et al.(1983) Gene 25:179-188) and the 3′ region of the nopalinesynthase gene from the T-DNA of the Ti plasmid of Agrobacteriumtumefaciens. The seed expression cassette comprising the phaseolin 5′region, the fragment encoding the Calendula conjugated fatty acidmodifying enzyme and the phaseolin 3′ region can be isolated as arestriction fragment. This fragment can then be inserted into a uniquerestriction site of the vector carrying the marker gene.

To 50 μL of a 60 mg/mL 1 mm gold particle suspension is added (inorder): 5 μL. DNA (1 μg/μL), 20 μL spermidine (0.1 M), and 50 μL CaCl₂(2.5 M). The particle preparation is then agitated for three minutes,spun in a microfuge for 10 seconds and the supernatant removed. TheDNA-coated particles are then washed once in 400 μL 70% ethanol andresuspended in 40 μL of anhydrous ethanol. The DNA/particle suspensioncan be sonicated three times for one second each. Five μL of theDNA-coated gold particles are then loaded on each macro carrier disk.

Approximately 300-400 mg of a two-week-old suspension culture is placedin an empty 60×15 mm petri dish and the residual liquid removed from thetissue with a pipette. For each transformation experiment, approximately5-10 plates of tissue are normally bombarded. Membrane rupture pressureis set at 1100 psi and the chamber is evacuated to a vacuum of 28 inchesmercury. The tissue is placed approximately 3.5 inches away from theretaining screen and bombarded three times. Following bombardment, thetissue can be divided in half and placed back into liquid and culturedas described above.

Five to seven days post bombardment, the liquid media may be exchangedwith fresh media, and eleven to twelve days post bombardment with freshmedia containing 50 mg/mL hygromycin. This selective media can berefreshed weekly. Seven to eight weeks post bombardment, green,transformed tissue may be observed growing from untransformed, necroticembryogenic clusters. Isolated green tissue is removed and inoculatedinto individual flasks to generate new, clonally propagated, transformedembryogenic suspension cultures. Each new line may be treated as anindependent transformation event. These suspensions can then besubcultured and maintained as clusters of immature embryos orregenerated into whole plants by maturation and germination ofindividual somatic embryos.

Using methods described in this Example, transformed soybean embryoswith detectable levels of conjugated polyunsaturated fatty acids may beidentified and propagated.

Example 8 Expression of Chimeric Genes in Microbial Cells

The cDNAs encoding the instant fatty acid modifying enzymes associatedwith conjugated double bond synthesis comprising the delta-9 position inseeds of Calendula officinalis can be inserted into the T7 E. coliexpression vector pET24d (Novagen). For example, plasmid DNA containinga cDNA may be appropriately digested to release a nucleic acid fragmentencoding the fatty acid modifying enzymes associated with conjugateddouble bond synthesis in seeds of Calendula officinalis. This fragmentmay then be purified on a 1% NuSieve GTG™ low melting agarose gel (FMC).Buffer and agarose contain 10 μg/ml ethidium bromide for visualizationof the DNA fragment. The fragment can then be purified from the agarosegel by digestion with GELase™ (Epicentre Technologies) according to themanufacturer's instructions, ethanol precipitated, dried and resuspendedin 20 μL of water. Appropriate oligonucleotide adapters may be ligatedto the fragment using T4 DNA ligase (New England Biolabs, Beverly,Mass.). The fragment containing the ligated adapters can be purifiedfrom the excess adapters using low melting agarose as described above.The vector pET24d is digested, dephosphorylated with alkalinephosphatase (NEB) and deproteinized with phenol/chloroform as describedabove. The prepared vector pET24d and fragment can then be ligated at16° for 15 hours followed by transformation into DH5 electrocompetentcells (GIBCO BRL). Transformants can be selected on agar platescontaining 2×YT media and 50 μg/mL kanamycin. Transformants containingthe gene are then screened for the correct orientation with respect topET24d T7 promoter by restriction enzyme analysis.

Clones in the correct orientation with respect to the T7 promoter can betransformed into BL21(DE3) competent cells (Novagen) and selected on2×YT agar plates containing 50 μg/ml kanamycin. A colony arising fromthis transformation construct can be grown overnight at 30° C. in 2×YTmedia with 50 μg/mL kanamycin. The culture is then diluted two fold withfresh media, allowed to re-grow for 1 h, and induced by addingisopropyl-thiogalactopyranoside to 1 mM final concentration. Cells arethen harvested by centrifugation after 3 h and re-suspended in 50 μL of50 mM Tris-HCl at pH 8.0 containing 0.1 mM DTT and 0.2 mM phenylmethylsulfonyl fluoride. A small amount of 1 mm glass, beads can beadded and the mixture sonicated 3 times for about 5 seconds each timewith a microprobe sonicator. The mixture is centrifuged and the proteinconcentration of the supernatant determined. One μg of protein from thesoluble fraction of the culture can be separated by SDS-polyacrylamidegel electrophoresis. Gels can be observed for protein bands migrating atthe expected molecular weight.

Example 9

Conjugated 18:3 Fatty Acids can Improve Carcass Quality when added toAnimal Feed

Experiments were conducted to evaluate the effects of feedingeleostearic (18:3) conjugated fatty acids on pig growth, carcasscharacteristics, and fat firmness. Twenty-four pigs (barrows, castratedmales, from PIC genetics) with a capacity for high rates of daily leangrowth and reduced back fat were randomly assigned by litter mates,weight, and block to three dietary treatments. Group one was fed normalcorn feed, group two received normal corn feed supplemented with CLA,and the third group received normal corn feed supplemented withconjugated linolenic acids, i.e., ClnAs (18:3 conjugated fatty acids).Pigs were penned individually and identified by ear tattoo. The averageinitial weight of the barrows was 125 pounds. Pigs were placed on theirrespective test diets at 150 lb, after being fed a common diet.

Diets were fed in two phases: Phase 1 (150 to 200 lb), and Phase 2 (200to 250 lb). Ingredient and nutrient compositions of the treatment dietsare shown in Table 4 and Table 5, respectively. The diets wereformulated to be isocaloric.

TABLE 4 Ingredient Composition of Diets Ingredient, % NC¹ NC + CLA NC +ClnA Grower Diets NC¹ 69.826 69.826 69.826 Soybean Meal, 48%² 25.28325.283 25.283 A-V Fat³ 2.498 2.498 2.498 L-Lysine-HCl⁴ 0.073 0.073 0.073Limestone⁵ 0.838 0.838 0.838 Dical 21⁶ 0.761 0.761 0.761 Choline CH,60%⁷ 0.096 0.096 0.096 TM & Vitamin Premix⁸ 0.250 0.250 0.250 Salt⁹0.300 0.300 0.300 Copper Sulfate¹⁰ 0.075 0.075 0.075 Finisher Diets NC75.142 75.142 75.142 Soybean Meal, 48% 20.340 20.340 20.340 A-V Fat2.564 2.564 2.564 Limestone 0.740 0.740 0.740 Dical 21 0.525 0.525 0.525Choline CH, 60% 0.065 0.065 0.065 TM & Vitamin Premix 0.250 0.250 0.250Salt 0.300 0.300 0.300 Copper Sulfate 0.075 0.075 0.075 ¹normal hybridcorn, W677, from Wyffels, Atkinson, IL ²Perdue Farms, Inc., Greenville,NC ³Moyer Packing Co., Souderton, PA ⁴Archer Daniels Midland Co.,Decatur, IL ⁵Akey, Inc. Lewisburg, OH ⁶Potash Company of Saskatchewan,Davenport, IA ⁷Akey, Inc. Lewisburg, OH ⁸Trace Minerals and VitaminPremix, Young's, Greensboro, MD ⁹Akey, Inc. Lewisburg, OH ¹⁰Akey, Inc.Lewisburg, OH

TABLE 5 Calculated nutrient composition of treatment diets. Phase 1Phase 2 Nutrient (150-200 lb) (200-250 lb) Energy, kcal/lb 1734 1756Energy, kcal/kg 3823 3871 Protein, mcal % 18.00 16.00 Lysine, mcal %1.05 0.86 Methionine + Cysteine, mcal % 0.64 0.61 Calcium, % 0.60 0.50Total Phosphorus 0.55 0.49

The mixer used to prepare the diets was flushed with 300 lb corn priorto mixing and between each mix to prevent cross-contamination.Conjugated linoleic acid (CLA) was purchased from Conlinco, Inc.(Detroit Lakes, Minn.) as “Clareen™”. Conjugated linolenic acid (ClnA)was from a commercial source of tung oil (Industrial Oil Products,Woodbury, N.Y.) that was approximately 65% α-eleostearic acid. Toachieve a final conjugated fatty acid concentration of 0.50%, 0.83 lbCLA preparation/100 lb diet and 0.73 lb CLnA preparation/100 lb of dietwere added. To minimize oxidation of the conjugated fatty acid, dietswere prepared each 14 days and refrigerated until use. Feed was added tofeeders in minimal amounts daily. The antibiotic bacitracin methylenedisalicylate (BMD, Alpharma, Inc., Fort Lee, N.J.) was included in alldiets (50 g/ton). Feed samples were collected for amino acid and fattyacid analysis.

Live weights were recorded to determine average daily gains Phase 1 (150to 200 lbs), and Phase 2 (200 to 250 lbs). Feed weight data were alsocollected to determine feed efficiency. Animals were observed 2-3 timesdaily for access to feeders and waterers, house temperatures, and anyabnormal health conditions. Pigs were not replaced during the trial. Anyanimals that died were necropsied to determine the cause of death. Deadanimal body weights were used to correct feed efficiency.

When pigs reached 250 pounds body weight they were slaughtered,processed and standard carcass measurements were collected. Because oflimitations on conjugated fatty acid, pigs fed CLA and CLnA were fed acommon diet four days prior to slaughter. Bellies from the eight pigs ineach study group were evaluated for fat firmness evaluated by measuringbelly thickness before and after compression. Fat compression wasachieved by placing a 50 lb weight on the fresh belly for one hour. Fatcompression was quantified by subtracting the compressed belly thicknessfrom the initial belly thickness. Belly thickness was measured using amicrometer. The results of the belly compression evaluation are shown inTable 6. Data were analyzed as a randomized complete block design usingthe GLM (General Linear Model) procedure of SAS (Statistical AnalysisSystems). Table values represent the difference between compressed anduncompressed pork belly thickness. A smaller number indicates reducedcompression (i.e. greater firmness) of pork bellies. Because a porkbelly is greater than 50% fat, the belly compression test is anindicator of relative firmness of pork belly fat. Addition of either CLAor ClnA to NC diets resulted in greater fat firmness in pigs. Theimproved pork fat firmness resulting from dietary addition of CLA isconsistent with results reported by others (Eggert, J. M., et al. (1999)J. Anim. Sci. 77(Suppl):53; Thiel, R. C., et al. (1998) J. Anim. Sci.76(Supp):13; Wiegand, B. R., F. C. Parrrish Jr and J. C. Sparks (1999)J. Anim. Sci. 77(Suppl):19; U.S. Pat. No. 5,554,646; and U.S. Pat. No.5,851,572). Improved fat firmness resulting from dietary CLnA inclusionhas not been previously reported. Based on the results of thisexperiment, addition of conjugated linoleic acid (CLA) or conjugatedlinolenic acid (ClnA) to pig diets results in improved fat firmness.

TABLE 6 Results of Fat Compression Test Measurement NC NC + CLA NC +CLnA SEM¹ Pork Belly Compression, 33.2² 28.0 30.8 0.68 mm ¹StandardError of the Mean ²All three test sample means were statisticallydifferent (P < 0.05)

Example 10 Production of Calendic Acid in Somatic Soybean Embryos

The Calendula officinalis clones CalFad2-1 and CalFad2-2 were expressedin somatic soybean embryos in order to examine their activity in a cropspecies.

The open-reading frames of these cDNAs were amplified by PCR in ordergenerate the appropriate flanking restriction enzyme sites for cloninginto the soybean expression vector. The oligonucleotide primers used foramplification of the CalFad2-1 open-reading frame were:5′-ttgcggccgcTACACCTAGCTACGTACCATG-3′ (sense, SEQ ID NO:18) and5′-ttgcggccgTCACGGTACTGATGATGGCAC-3′ (antisense, SEQ ID NO:19). Theoligonucleotide primers used for amplification of the CalFad2-2 codingsequence were: 5′-agcggccgcTATACCATGGGCAAG-3′ (sense, SEQ ID NO:20) and5′-tgcggccgcTATGTTAAACTTC-3′ (antisense, SEQ ID NO:21). [Note: Thesequences shown in lower case contain an added NotI site along withadditional bases to facilitate restriction enzyme digestion.] Thetemplate was the cDNA corresponding to either EST ecs1c.pk009.n14(CalFad2-1) or EST ecs1c.pk008.a24 (CalFad2-2), and Pfu polymerase(Stratagene) was used for the amplification reactions. The resulting PCRproducts were subcloned into the intermediate vector pCR-Script AMPSK(+) (Stratagene) according to the manufacturer's protocol. Theamplified CalFad2-1 and CalFad2-2 coding sequences were then releasedwith NotI digestion and then subcloned into the corresponding site ofthe soybean expression vector pKS67.

Vector pKS67 contains the promoter of the gene for the α′ subunit ofβ-conglycinin [Beachy et al., (1985) EMBO J. 4:3047-3053], which allowsfor strong seed-specific expression of transgenes. This vector wasconstructed as follows. A plasmid pZBL100 containing chimeric genes toallow expression of hygromycin B phosphotransferase in certain bacteriaand in plant cells was constructed from the following genetic elements:a.) T7 promoter+Shine-Delgarno/hygromycin B phosphotransferase (HPT)/T7terminator sequence, b.) 35S promoter from cauliflower mosaic virus(CaMV)/hygromycin B phosphotransferase (HPT)/nopaline synthase (NOS3′from Agrobacterium tumefaciens T-DNA, and c.) pSP72 plasmid vector(Promega) with β-lactamase coding region (ampicillin resistance gene)removed.

The hygromycin B phosphotransferase gene was amplified by PCR from E.coli strain W677 (Gritz, L. and Davies, J (1983) Gene 25:179-188 whichcontained a Klebsiella derived plasmid pJR225 (Gritz, L. and Davies, J(1983) Gene 25:179-188. Starting with the pSP72 vector (Promega) theelements were assembled into a single plasmid using standard cloningmethods (Maniatis).

Plasmid pZBL100 thus contains the T7 promoter/HPT/T7 terminator cassettefor expression of the HPT enzyme in certain strains of E. coli, such asNovaBlue (DE3) (Novagen), that are lysogenic for lambda DE3 (whichcarries the T7 RNA Polymerase gene under lacUV5 control). PlasmidpZBL100 also contains the 35S/HPT/NOS cassette for constitutiveexpression of the HPT enzyme in plants, such as soybean. These twoexpression systems allow selection for growth in the presence ofhygromycin to be used as a means of identifying cells that contain theplasmid in both bacterial and plant systems.

PZBL100 also contains three unique restriction endonuclease sitessuitable for the cloning of other chimeric genes into this vector.

The construction of a plasmid for expression of the CalFad2-1 andCalFad2-2 coding sequences under control of the soybean β-conglycinin α′subunit promoter (Beachy et al., (1985) EMBO J. 4:3047-3053) wasfacilitated by the use of plasmids pCW109 and pML18, both of which havebeen described (see World Patent Publication No. WO94/11516).

A unique NotI site was introduced into the cloning region between theβ-conglycinin promoter and the phaseolin 3′ end in pCW109 by digestionwith NcoI and XbaI followed by removal of the single stranded DNA endswith mung bean exonuclease. NotI linkers (New England Biolabs) wereligated into the linearized plasmid to produce plasmid pAW35. The singleNotI site in pML18 was destroyed by digestion with NotI, filling in thesingle stranded ends with dNTPs and Klenow fragment followed byre-ligation of the linearized plasmid. The modified pML18 was thendigested with HindIII and treated with calf intestinal phosphatase.

The β-conglycinin:NotI:phaseolin expression cassette in pAW35 wasremoved by digestion with Hind III and the 1.8 kB fragment was isolatedby agarose gel electrophoresis. The isolated fragment was ligated intothe modified and linearized pML18 construction described above. A clonewith the desired orientation was identified by digestion with NotI andXbaI to release a 1.08 kB fragment indicating that the orientation ofthe β-conglycinin transcription unit was the same as the selectablemarker transcription unit. The resulting plasmid was given the namepBS19.

HindIII is one of the unique cloning sites available in pZBL100. Toassemble the final expression cassette, pBS19 and pZBL100, were bothdigested with HindIII. The β-conglycinin containing fragment from pBS19was isolated by gel electrophoresis and ligated into the digestedpZBL100, which had been treated with calf alkaline phosphatase. Theresulting plasmid was named pKS67.

The PCR amplified coding sequences of CalFad2-1 and CalFa2-2 were fusedwith the β-conglycinin promoter and phaseolin termination sequences invector pKS67 was transformed into somatic soybean embryos as follows. Toinduce somatic embryos, cotyledons, 3-5 mm in length dissected fromsurface sterilized, immature seeds of a soybean cultivar A2872 orJACK-910 were cultured in the light or dark at 26° C. on an appropriateagar medium for 6-10 weeks. Somatic embryos that produce secondaryembryos were then excised and placed into a suitable liquid medium.After repeated selection for clusters of somatic embryos that multipliedas early, globular staged embryos, the suspensions were maintained asdescribed below.

Soybean embryogenic suspension cultures were maintained in 35 mL liquidmedia on a rotary shaker, 150 rpm, at 26° C. with florescent lights on a16:8 hour day/night schedule. Cultures were subcultured every two weeksby inoculating approximately 35 mg of tissue into 35 mL of liquidmedium.

Soybean embryogenic suspension cultures were then transformed with thevector pKS67 containing the coding sequence for CalFad2-1 and CalFad2-2by the method of particle gun bombardment (Klein et al. (1987) Nature(London) 327:70, U.S. Pat. No. 4,945,050). A Du Pont BiolisticaPDS1000/HE instrument (helium retrofit) was used for thesetransformations.

To 50 mL of a 60 mg/mL 1 mm gold particle suspension were added (inorder): 5 mL DNA (1 mg/mL), 20 ml spermidine (0.1 M), and 50 mL CaCl₂(2.5 M). The particle preparation was then agitated for three minutes,spun in a microfuge for 10 seconds and the supernatant removed. TheDNA-coated particles were then washed once in 400 mL 70% ethanol andresuspended in 40 mL of anhydrous ethanol. The DNA/particle suspensionwas sonicated three times for one second each. Five mL of the DNA-coatedgold particles was then loaded on each macro carrier disk.

Approximately 300-400 mg of a two-week-old suspension culture was placedin an empty 60×5-mm petri dish and the residual liquid removed from thetissue with a pipette. For each transformation experiment, approximately5 to 10 plates of tissue were bombarded. Membrane rupture pressure wasset at 1100 psi and the chamber was evacuated to a vacuum of 28 inchesmercury. The tissue was placed approximately 3.5 inches away from theretaining screen and bombarded three times. Following bombardment, thetissue was divided in half and placed back into liquid and cultured asdescribed above.

Five to seven days post bombardment, the liquid media was exchanged withfresh media, and eleven to twelve days post bombardment with fresh mediacontaining 50 mg/mL hygromycin. This selective media was refreshedweekly. Seven to eight weeks post bombardment, green, transformed tissuewas observed growing from untransformed, necrotic embryogenic clusters.Isolated green tissue was removed and inoculated into individual flasksto generate new, clonally propagated, transformed embryogenic suspensioncultures. Each new line was treated as an independent transformationevent. These suspensions were then subcultured and maintained asclusters of immature embryos. Immature embryos at this stage producestorage products, including storage lipids that are similar incomposition to zygotic embryos at a similar stage of development (seeWorld Patent Publication No. WO94/11516).

Transgenic soybean embryos selected and maintained in this manner wereanalyzed for calendic acid content using gas chromatography (GC) or gaschromatography-mass spectrometry (GC-MS). Individual embryos expressingeither CalFad2-1 or CalFad2-2 were homogenized in 1% (w/v) sodiummethoxide in methanol. Fatty acid methyl esters resulting from thistransesterification step were analyzed by GC and GC-MS using methodsdescribed in Example 3. In somatic embryos expressing either cDNA, afatty acid methyl ester with retention time and mass spectrum equivalentto that of methyl calendic acid from Calendula officinalis seed extractswas detected (FIG. 5). This fatty acid methyl ester was not detected inextracts from untransformed embryos. To further confirm the identity ofmethyl calendic acid in soybean embryos expressing CalFad2-1 andCalFad2-2, fatty acid methyl; esters from the transgenic tissue wasreacted with 4-methyl-1,2,4-triazoline-3,5-dione (MTAD) [Dobson, G.(1998) J. Am. Oil Chem. Soc. 75:137-142] and the resulting derivativeswere analyzed by GC-MS. (This reagent reacts primarily with conjugatedtrans-double bonds to form Diels-Alder adducts with MTAD.) AHewlett-Packard 6890 GC linked to a Hewlett-Packard 5973 mass selectivedetector was used for these analyses. Samples were resolved with aDB1-Ht column (15 m×0.25 mm I.D.) (J&W Scientific), and the oventemperature was programmed from 185° C. (3-min hold) to 285° C. at arate of 2° C./min. The mass spectrum of the MTAD derivative of the novelfatty acid methyl ester in the transgenic soybean embryos expressingCalFad2-1 and CalFad2-2 was identical to that of the MTAD derivative ofmethyl calendic acid from Calendula officinalis seed extracts (FIG. 6).These mass spectra were characterized by a molecular ion of 405 m/z andalso by a major diagnostic ion of 262 m/z. These results thus confirmthat somatic soybean embryos expressing either CalFad2-1 or CalFad2-2produce calendic acid.

Table 7 shows a comparison of the fatty acid compositions ofuntransformed somatic soybean embryos and embryos from transgenic linesMSE 284-2-6 and MSP 425-12-2 that are transformed with the CalFad2-1 andCalFad2-2 cDNAs, respectively, behind the seed-specific β-conglycinin α′subunit promoter (as described above).

TABLE 7 Somatic Embryo Fatty Acid Compositions From Soybean TransgenicLines MSE 284-2-6 and MSP 425-12-2 Expressing the CalFad2-1 andCalFad2-2 cDNAs Associated with Calendic Acid Synthesis MSE 284-2-6 MSP425-12-2 Fatty Acid Untransformed (CalFad2-1) (CalFad2-2) Weight %¹: (n= 5)² (n = 5) (n = 5) 16:0 14.2 ± 0.8 12.2 ± 1.0 12.3 ± 1.1 18:0  2.9 ±0.4  3.5 ± 0.9  3.3 ± 0.5 18:1  7.2 ± 1.0  9.0 ± 1.5  8.6 ± 2.1 18:253.5 ± 3.2 52.2 ± 2.4 38.2 ± 3.4 18:3 20.6 ± 2.5 18.8 ± 3.0 17.7 ± 3.5Calendic Acid N.D.³  3.1 ± 1.0 18.1 ± 3.6 Other ≦1.6 ≦1.3 ≦1.7 ¹Thefatty acid compositions are given as the weight percentage of totalfatty acids of somatic soybean embryos measured by gas chromatography asdescribed above. ²Values were obtained from five separate measurements(± standard deviation) of single embryos ³N.D., Not detected.

Example 11 Production of Dimorphecolic Acid in Transgenic SomaticSoybean Embryos

The seed oil of Dimorphotheca species including Dimorphotheca sinuata isenriched in the unusual C₁₈ fatty acid dimorphecolic acid(9-OH-18:2Δ^(10trans, 12trans)), which contains two conjugatedtrans-double bonds between the Δ¹⁰ and Δ¹¹ and between the Δ¹² and Δ¹³carbon atoms as well as a hydroxyl group on the Δ⁹ carbon atom [Binder,R. G. et al., (1964) J. Am. Oil Chem. Soc. 41:108-111; Morris, L. J. etal., (1960) J. Am. Oil Chem. Soc. 37:323-327]. From the resultsdescribed below, it is believed that dimorphecolic acid is produced in abiosynthetic pathway involving the activities of two diverged forms ofthe Δ¹²-oleic acid desaturase (Fad2) from Dimorphotheca, designatedDMFad2-1 and DMFad2-2 (FIG. 7). Expression data from transgenic somaticsoybean embryos (as described below) is consistent with DMFad2-1catalyzing the formation of a Δ^(12trans) double-bond in oleic acid toform 18:2Δ^(9cis, 12trans). The Δ^(9cis) double bond of this fatty acidintermediate is then modified by DMFad2-2 to form 9-OH and a Δ^(10trans)double bond. Therefore, the hydroxylation of the Δ⁹-position by DMFad2-2leads to the formation of conjugated double-bonds. The product of thesetwo steps is dimorphecolic acid.

The cDNAs for DMFad2-1 and DMFad2-2 were derived from ESTs for divergedFad2s that were identified among pools of ESTs from a Dimorphothecasinuata seed cDNA library (dms2c.pk006.d7, SEQ ID NO:10; anddms2c.pk001.I13, SEQ ID NO:12). It is notable that the amino acidsequence corresponding to DMFad2-2 (SEQ ID NO:13) is most related tothose of CalFad2-1 (SEQ ID NO: 2) and CalFad2-2 (SEQ ID NO: 4), whichhave been demonstrated to catalyze the formation of conjugated doublebonds by modification of the delta-9 position of linoleic acid (Examples3, 4, and 10). Using the multiple sequence comparison program Megalign(v3.1.7) from the Lasergene□ software package (DNASTAR Inc., Madison,WI) and the Clustal method of alignment (default program parameters),the amino acid sequence of DMFad2-2 (SEQ ID NO:13) shares 72.5% identitywith the amino acid sequence of CalFad2-1 (SEQ ID NO:2) and 74.0%identity with the amino acid sequence of CalFad2-2 (SEQ ID NO:4). Incontrast, the amino acid sequence of DMFad2-2 (SEQ ID NO: 3) shares lessthan 52% identity with any of the other Fad2-related polypeptides shownin FIGS. 2A-2D including those of the castor hydroxylase andconventional ω⁶-oleic acid desaturases like the soybean, borage, andsunflower polypeptides. Therefore, given its close relation to CalFad2-1and CalFad2-2, it is believed that DMFad2-2 is associated with themodified delta-9 position that is present in dimorphecolic acid. It isalso notable that the residue immediately adjacent to the firsthistidine box in the amino acid sequences of DMFad2-1 and DMFad2-2 is aglycine (as indicated by an asterisk in FIGS. 2A-2D). A glycine in thisposition is only observed in ω⁶-oleic acid desaturase-related enzymesthat have diverged functionality, such as the castor oleic acidhydroxylase (van de Loo, F. J. et al. (1995) Proc. Natl. Acad. Sci.U.S.A. 92:6743-6747) and the Crepis palaestina epoxidase (Lee, M. et al.(1998) Science 280:915-918). Given this feature of their primarystructures, it is believed that the polypeptides encoded by DMFad2-1 andDMFad2-2 are associated with dimorphecolic acid synthesis and are notconventional ω⁶-oleic acid desaturases involved in standard fatty acidsynthesis.

Initially, the open-reading frames of cDNAs for DMFad2-1 and DMFad2-2were amplified by PCR using Pfu polymerase (Stratagene) to generate theappropriate restriction enzyme sites for cloning into the soybeanexpression vector. For amplification of the open-reading frame of theDMFad2-1 cDNA, EST dms2c.pk006.d7 (SEQ ID NO:10 for the nucleotidesequence, and SEQ ID NO:11 for the polypeptide translation product) wasused as the template, and the oligonucleotide primers were:5′-tatgcggccgcAAATGGGAGCAGGAGGTTG-3′ (sense, SEQ ID NO:22) and5′-tttgcggccgcATTACATCTTATTCTTGTACC-3′ (antisense, SEQ ID NO:23). Foramplification of the open-reading frame of the DMFad2-2 cDNA, ESTdms2c.pk001.I13 (SEQ ID NO:12 for the nucleotide sequence, and SEQ IDNO:13 for the polypeptide translation product) was used as the template,and the oligonucleotide primers were:5′-tgcggccgcAATGGGTGGAGGGATGGGAGCATCTGAG-3′ (sense, SEQ ID NO:24) and5′-tagcggccgcTGATTAATCAAGTCTTAG-3′ (antisense, SEQ ID NO:25). Thenucleotides shown in lower case are not Dimorphotheca sequences, butinstead encode an added NotI site along with additional bases tofacilitate restriction enzyme digestion. The resulting PCR products weresubcloned into the intermediate vector pCR-Script AMP SK(+) (Stratagene)according to the manufacturer's protocol. The DMFad2-1 and DMFad2-2 PCRproducts were then moved as NotI fragments into corresponding site ofthe soybean expression vector pKS67 behind the promoter of the gene forthe α′ subunit of β-conglycinin. The construction of vector pKS67 isdescribed in Example 10. The DMFad2-1 NotI fragment was also subclonedinto the soybean expression vector pKS17, which is equivalent to vectorpKS67 except that it lacks the 35S/hygromycin phosphotransferase(HPT)INOS cassette for constitutive expression of the HPT enzyme inplants.

The expression constructs containing the DMFad2-1 and DMFad2-2 codingsequences in vector pKS67 were transformed into somatic soybean embryosusing the biolistic method as described in Example 10. To determinetheir functions, DMFad2-1 and DMFad2-2 were expressed individually orco-expressed in somatic soybean embryos. The fatty acid compositions ofthe resulting transgenic soybean embryos were then assessed for thepresence of novel fatty acid structures. The individual transformationexperiments were MSE331 (DMFad2-1) and MSE229 (DMFad2-2). Theco-expression transformation experiment (MSE 330) was conducted in whichthe DMFad2-2 coding sequence in vector pKS67 was co-transformed with theDMFad2-1 coding sequence in vector pKS17 in somatic soybean embryos,using a molar ratio of 1:10 of the two expression constructs for thetransformation (using methods described in Example 10). The resultingtransgenic soybean embryos selected for hygromycin resistance wereanalyzed for alterations in fatty acid content relative to untransformedembryos. Fatty acid methyl esters were prepared by homogenization ofuntransformed and transgenic somatic soybean embryos in 1% (w/v) sodiummethoxide in methanol using methods described by Hitz et al. (1994)Plant Physiol. 105:635-641. Fatty acid methyl esters were dried undernitrogen and reacted with 50-100 μl of the silylating reagentbis(trimethylsilyl)trifluoroacetamide:trimethylchlorosilane (99:1 v/v)(Supelco) in order to convert the hydroxyl group of dimorphecolic acidto a trimethylsilyl (TMS) ether derivative for gas chromatography (GC)and gas chromatography-mass spectrometry (GC-MS) analysis. The recoveredfatty acid methyl esters and derivatives were then analyzed using aHewlett-Packard 6890 chromatograph fitted with an Omegawax 320 column(30 m×0.32 mm inner diameter; Supelco). The oven temperature wasprogrammed from 185° C. (4 min hold) to 215° C. at a rate of 5° C./minand then to 240° C. at 20° C./min (1 min hold). Fatty acid methyl esterswere also analyzed by GC-MS an HP6890 interfaced with a HP5973(Hewlett-Packard) mass selective detector. Compounds were resolved usingan HP-INNOWax column (30 m×0.25 mm inner diameter) with the oventemperature programmed from 180° C. (3.5-min hold) to 215° C. at a rateof 2° C./min (2-min hold) and then to 230° C. at 10° C./min.

GC analysis of fatty acid methyl esters from transgenic soybean embryosexpressing DMFad2-1 (transformation experiment MSE 331) indicated thepresence of a peak that eluted immediately after methyl linoleic acid(18:2Δ^(9cis, 12cis)) (FIG. 8), but was absent from untransformedembryos. This peak had the same retention time and mass spectrum as themethyl ester of 18:2Δ^(9cis, 12trans) that is found in the developingDimorphotheca sinuata seeds. The novel fatty acid resulting fromDMFad2-1 expression in transgenic soybean embryos is thus identified as18:2Δ^(9cis, 12trans). Amounts of this fatty acid measured in singlesoybean embryos from experiment MSE 331 ranged from 0 to 21 wt % of thetotal fatty acids. Based on these results, DMFad2-1 was identified as afatty acid modifying enzyme associated with the synthesis of thetrans-Δ¹² double bond of 18:2Δ^(9cis, 12trans).

Fatty acid methyl esters from transgenic soybean embryos expressingDMFad2-2 (transformation experiment MSE 229) were analyzed by GC-MSusing a selected ion scan for ion 225 m/z, which is the most abundantion in the mass spectrum of the TMS derivative of methyl dimorphecolicacid. In these selected ion chromatograms, two peaks were detected thatdisplayed mass spectra equivalent to that of the TMS derivative ofmethyl dimorphecolic acid. The less abundant of these peaks had the sameretention time as that of the TMS derivative of methyl dimorphecolicacid from Dimorphotheca sinuata seeds. However, the larger of the twopeaks displayed a shorter retention time and is tentatively identifiedas the cis-Δ¹² isomeric form of dimorphecolic acid(9-OH-18:2Δ^(9cis, 12cis)), which has previously been reported to occurin trace amounts in the seed oil of Dimorphotheca species [Morris, L.J., et al., (1960) J. Am. Oil Chem. Soc. 37:323-327]. This isomeric formof dimorphecolic acid likely arises from the modification of the Δ⁹double bond of linoleic acid (18:2Δ^(9cis, 12cis)) by DMFad2-2. Thedimorphecolic acid isomers detected in soybean embryos expressingDMFad2-2 accounted for <0.1% of the total fatty acids.

Results from expression of DMFad2-2 alone suggested that a limitingfactor in the synthesis of dimorphecolic acid is the lack of asignificant substrate pool of the trans-Δ¹² isomer of linoleic acid(18:2Δ^(9cis, 12trans)) in somatic soybean embryos. As described above,this fatty acid is the product of DMFad2-1. Therefore, in an attempt toincrease amounts of dimorphecolic acid in transgenic embryos, DMFad2-1and DMFad2-2 were co-expressed in somatic soybean embryos(transformation experiment MSE 330). The resulting embryos accumulatedboth 18:2Δ^(9cis, 12trans) and the predominant form of dimorphecolicacid (9-OH-18:2Δ^(9cis, 12trans)) which are also found in the seed oilof Dimorphotheca sinuata (FIG. 9 and 10). In these embryos,dimorphecolic acid accounted for 0.5 to 1 wt % of the total fatty acidscompared to accumulations of less than 0.1% in “DMFad2-2 alone”transformants. In addition, the primary form of dimorphecolic aciddetected in the “DMFad2-2 alone” transformations was the tentativelyidentified as the Δ^(12cis) isomer of dimorphecolic acid(9-OH-18:2Δ^(9cis, 12cis), see FIGS. 7 and 9). These results are thusconsistent with a biosynthetic pathway for dimorphecolic acid involvingthe initial activity of DMFad2-1 that generates 18:2Δ^(9cis, 12trans).The Δ⁹ double bond of this fatty acid is then modified to a 9-hydroxyand Δ^(10trans) double bond by DMFad2-2 to yield dimorphecolic acid(FIG. 7).

Example 12 Characterization of the Trans-Linoleic Product of DMFad2-1

To further characterize the structure of the putative18:2×^(9cis, 12trans) isomer from soybean embryos expressing DMFad2-1,the methyl ester of this fatty acid is purified to near homogeneity fromthe transgenic embryos and analyzed by ¹H-¹³C NMR two-dimensionalcorrelation NMR. The methyl ester of the putative 18:2Δ^(9cis, 12trans)is purified from extracts of transgenic soybean embryos using acombination of reverse-phase and argentation thin layer chromatography(TLC). Fatty acid methyl esters from soybean embryos expressing DMFad2-1are initially resolved by reverse phase TLC using a solvent system ofmethanol:acetonitrile:water (60:40:1 v/v/v) and 20 cm×20 cm RP18 TLCplates (Merck). TLC plates containing the crude fatty acid methyl estersare developed sequentially to 10-cm, 15-cm and finally to thefull-length of the plate. TLC plates are dried under nitrogen betweendevelopments. A band containing a mixture of methyl 18:2Δ^(9cis, 12cis)and putative 18:2Δ^(9cis, 12trans) is identified by light staining withiodine vapor and then recovered from the scraped TLC matrix usinghexane:isopropanol (7:2 v/v). These two isomers are then resolved usingargentation TLC with 10-cm×20-cm silica gel K60 plates (Whatman) thatare saturated with a solution of 5% (w/v) silver nitrate inacetonitrile. The methyl 18:2Δ^(9cis, 12cis) and putative18:2Δ^(9cis, 12trans) isomers are then separated on the argentationplates using a solvent system of hexane:ethyl ether (80:20 v/v) andsequential developments as described above. The putative methyl18:2Δ^(9cis, 12trans) isomer, which displays a higher mobility thanmethyl 18:2Δ^(9cis, 12cis), is identified by ultraviolet absorbanceafter spraying the plate with 0.1% (w/v) 2,7-dichlorofluorescein inmethanol. The putative methyl 18:2Δ^(9cis, 12trans) isomer is thenrecovered from the scraped TLC matrix with hexane:ethyl ether (50:50v/v), and residual dichlorofluorescein is removed by washing the organiclayer with 1 M Tris (pH9.0). The sample is finally passed over a silicacolumn and eluted with, hexane:ethyl ether (80:20 v/v) to remove anyimpurities.

The purified putative methyl 18:2Δ^(9cis, 12trans) isomer derived fromthe transgenic soybean embryos is then analyzed by ¹H-¹³Ctwo-dimensional correlation NMR. The spectrum shows vinyl proton(protons associated with carbon double-bonds) chemical shifts thatdiffer when the protons are in the cis versus trans orientation. Forinstance, vinyl proton chemical shifts from a methyl 18:2Δ^(9cis, 12cis)standard are 5.396, 5.380, 5.348, and 5.340 ppm (one reading for eachproton in the two double bonds), compared to a methyl18:2Δ^(9trans, 12trans) standard that has chemical shifts of 5.400,5.392, 5.434, and 5.434 ppm. The fatty acids from transgenic soybean areanalyzed in a comparable experiment and compared to the known methyl18:2Δ^(9cis, 12trans) isomer isolated form Dimorphotheca seed oil(Morris, et al., (1960) J. Am. Oil Chem. Soc. 37:323-327; Morris andMarshall (1966) Chem & Ind 1493-1494).

25 1 1457 DNA Calendula officinalis CDS (31)..(1152) 1 aaaccactatactacaccta gctacgtacc atg ggc aaa gga gca tca aac aag 54 Met Gly Lys GlyAla Ser Asn Lys 1 5 aag gtt ttg gaa cga gtt cca atc aca aaa ccg cca ttcgaa tac aat 102 Lys Val Leu Glu Arg Val Pro Ile Thr Lys Pro Pro Phe GluTyr Asn 10 15 20 gat ctg aag aaa gca gta cca cca cat tgt ttt tca cga ccactt ttc 150 Asp Leu Lys Lys Ala Val Pro Pro His Cys Phe Ser Arg Pro LeuPhe 25 30 35 40 cgt tcg ttt tat ttc cta ctt cac gac att att gta aca tgtatc ctt 198 Arg Ser Phe Tyr Phe Leu Leu His Asp Ile Ile Val Thr Cys IleLeu 45 50 55 ttc tac gta gca tca aac tac att cct atg ctc cct ggt ttc ctttcc 246 Phe Tyr Val Ala Ser Asn Tyr Ile Pro Met Leu Pro Gly Phe Leu Ser60 65 70 tac att gta tgg cct gtt tac tgg atc tcc caa gga gtt ttt ctt ggc294 Tyr Ile Val Trp Pro Val Tyr Trp Ile Ser Gln Gly Val Phe Leu Gly 7580 85 aga ttg tgg atg att ggc cat gaa tgc ggc cat cat agt ttt agt aat342 Arg Leu Trp Met Ile Gly His Glu Cys Gly His His Ser Phe Ser Asn 9095 100 tac cgt tgg gtc gac gat agt gtt ggt ttt tta atc cat acg gcc acc390 Tyr Arg Trp Val Asp Asp Ser Val Gly Phe Leu Ile His Thr Ala Thr 105110 115 120 ctc act ccc tat ttt tcc ttc aaa tat agt cac cgt aat cac catgca 438 Leu Thr Pro Tyr Phe Ser Phe Lys Tyr Ser His Arg Asn His His Ala125 130 135 cac acc aat tcc atg gaa tat gac gaa gtt cat atc ccg aaa cgcaaa 486 His Thr Asn Ser Met Glu Tyr Asp Glu Val His Ile Pro Lys Arg Lys140 145 150 tcc gaa gct cta gat ctc tac ttt gaa ttt ctc ggc aac aac ccgatg 534 Ser Glu Ala Leu Asp Leu Tyr Phe Glu Phe Leu Gly Asn Asn Pro Met155 160 165 ggg tta atg atc acc atg tta tgt aaa ctc act ttt gga tat gcagct 582 Gly Leu Met Ile Thr Met Leu Cys Lys Leu Thr Phe Gly Tyr Ala Ala170 175 180 tac att atg ttc aat tat aca ggc aag aag cac aaa tct ggg ggttta 630 Tyr Ile Met Phe Asn Tyr Thr Gly Lys Lys His Lys Ser Gly Gly Leu185 190 195 200 gca agt cac ttc tac cca caa agc cct ctc ttt aac gac agcgaa cgt 678 Ala Ser His Phe Tyr Pro Gln Ser Pro Leu Phe Asn Asp Ser GluArg 205 210 215 aat cat gtt ttg ttc tct gat gtc ggg att tgc atc gtc ttgtac gca 726 Asn His Val Leu Phe Ser Asp Val Gly Ile Cys Ile Val Leu TyrAla 220 225 230 tgt tac cgt att gtg atg gtc aca ggg gca atg tcg gca ttttat gtg 774 Cys Tyr Arg Ile Val Met Val Thr Gly Ala Met Ser Ala Phe TyrVal 235 240 245 tac ggc att cct tgg gtt ata atg agt gct att ctc ttt gcagca act 822 Tyr Gly Ile Pro Trp Val Ile Met Ser Ala Ile Leu Phe Ala AlaThr 250 255 260 tat tta caa cac act cat cct tcg atc cct cat tat gat acaact gag 870 Tyr Leu Gln His Thr His Pro Ser Ile Pro His Tyr Asp Thr ThrGlu 265 270 275 280 tgg aac tgg ctt aga ggg gca tta tcg aca att gat agagat tta ggg 918 Trp Asn Trp Leu Arg Gly Ala Leu Ser Thr Ile Asp Arg AspLeu Gly 285 290 295 ttc ttc aac atg aac aaa aca cat tat cat gtt atc caccat tta ttt 966 Phe Phe Asn Met Asn Lys Thr His Tyr His Val Ile His HisLeu Phe 300 305 310 cct gtc att ccg gaa tac cat gca caa gag gca act gaggcc atc aag 1014 Pro Val Ile Pro Glu Tyr His Ala Gln Glu Ala Thr Glu AlaIle Lys 315 320 325 ccc atc tta ggt caa tat tac aag tat gat ggt act ccgttt tta aag 1062 Pro Ile Leu Gly Gln Tyr Tyr Lys Tyr Asp Gly Thr Pro PheLeu Lys 330 335 340 gcg ttg tgg aga gaa atg aag gac tgt att tat gta gaatcc gat caa 1110 Ala Leu Trp Arg Glu Met Lys Asp Cys Ile Tyr Val Glu SerAsp Gln 345 350 355 360 ggt cag aag aaa caa ggt att tac tgg ttc aag aataag att 1152 Gly Gln Lys Lys Gln Gly Ile Tyr Trp Phe Lys Asn Lys Ile 365370 tgaagtttca aataatctgg actacgttta attttgtgcc atcatcagta ccgtgaatta1212 gttttgttgt ggagagaaat gaaggactgt atttatgtag aatccgatca aggtcagaag1272 aaacaaggta tttactggtt caagaataag atttgaagtt tcaaataatc tggactacgt1332 ttaattttgt gccatcatca gtaccgtgaa ttagttttgt tgtgttttta attttaattt1392 cgtgtgatgg tgtaatgtaa tataattcag tataataaag gcgttatcct ttcatgggtt1452 taaaa 1457 2 374 PRT Calendula officinalis 2 Met Gly Lys Gly AlaSer Asn Lys Lys Val Leu Glu Arg Val Pro Ile 1 5 10 15 Thr Lys Pro ProPhe Glu Tyr Asn Asp Leu Lys Lys Ala Val Pro Pro 20 25 30 His Cys Phe SerArg Pro Leu Phe Arg Ser Phe Tyr Phe Leu Leu His 35 40 45 Asp Ile Ile ValThr Cys Ile Leu Phe Tyr Val Ala Ser Asn Tyr Ile 50 55 60 Pro Met Leu ProGly Phe Leu Ser Tyr Ile Val Trp Pro Val Tyr Trp 65 70 75 80 Ile Ser GlnGly Val Phe Leu Gly Arg Leu Trp Met Ile Gly His Glu 85 90 95 Cys Gly HisHis Ser Phe Ser Asn Tyr Arg Trp Val Asp Asp Ser Val 100 105 110 Gly PheLeu Ile His Thr Ala Thr Leu Thr Pro Tyr Phe Ser Phe Lys 115 120 125 TyrSer His Arg Asn His His Ala His Thr Asn Ser Met Glu Tyr Asp 130 135 140Glu Val His Ile Pro Lys Arg Lys Ser Glu Ala Leu Asp Leu Tyr Phe 145 150155 160 Glu Phe Leu Gly Asn Asn Pro Met Gly Leu Met Ile Thr Met Leu Cys165 170 175 Lys Leu Thr Phe Gly Tyr Ala Ala Tyr Ile Met Phe Asn Tyr ThrGly 180 185 190 Lys Lys His Lys Ser Gly Gly Leu Ala Ser His Phe Tyr ProGln Ser 195 200 205 Pro Leu Phe Asn Asp Ser Glu Arg Asn His Val Leu PheSer Asp Val 210 215 220 Gly Ile Cys Ile Val Leu Tyr Ala Cys Tyr Arg IleVal Met Val Thr 225 230 235 240 Gly Ala Met Ser Ala Phe Tyr Val Tyr GlyIle Pro Trp Val Ile Met 245 250 255 Ser Ala Ile Leu Phe Ala Ala Thr TyrLeu Gln His Thr His Pro Ser 260 265 270 Ile Pro His Tyr Asp Thr Thr GluTrp Asn Trp Leu Arg Gly Ala Leu 275 280 285 Ser Thr Ile Asp Arg Asp LeuGly Phe Phe Asn Met Asn Lys Thr His 290 295 300 Tyr His Val Ile His HisLeu Phe Pro Val Ile Pro Glu Tyr His Ala 305 310 315 320 Gln Glu Ala ThrGlu Ala Ile Lys Pro Ile Leu Gly Gln Tyr Tyr Lys 325 330 335 Tyr Asp GlyThr Pro Phe Leu Lys Ala Leu Trp Arg Glu Met Lys Asp 340 345 350 Cys IleTyr Val Glu Ser Asp Gln Gly Gln Lys Lys Gln Gly Ile Tyr 355 360 365 TrpPhe Lys Asn Lys Ile 370 3 1311 DNA Calendula officinalis CDS(39)..(1154) 3 aggaattcgg caccagccaa aaccaaagcc actatacc atg ggc aag gcagca tca 56 Met Gly Lys Ala Ala Ser 1 5 gcc aag aag gtt ttg gag cga gttcca atc tca aaa ccg cca ttc gaa 104 Ala Lys Lys Val Leu Glu Arg Val ProIle Ser Lys Pro Pro Phe Glu 10 15 20 tac aat gat ctg aag aaa gca gta ccacca cat tgt ttt tca cga cca 152 Tyr Asn Asp Leu Lys Lys Ala Val Pro ProHis Cys Phe Ser Arg Pro 25 30 35 ctt tcc cga tcc ttg tat ttc ctc ttt cacgac att att gta aca tgt 200 Leu Ser Arg Ser Leu Tyr Phe Leu Phe His AspIle Ile Val Thr Cys 40 45 50 atc ctt ttc tac gta gca tca aac tac att catatg ctc cct cgt ttc 248 Ile Leu Phe Tyr Val Ala Ser Asn Tyr Ile His MetLeu Pro Arg Phe 55 60 65 70 ctt tcc tgc atc gta tgg cct gtt tac tgg atctcc caa gga gtt ttt 296 Leu Ser Cys Ile Val Trp Pro Val Tyr Trp Ile SerGln Gly Val Phe 75 80 85 ctc ggc aga ttg tgg atg atc ggc cac gaa tgc ggtcat cat agc ttc 344 Leu Gly Arg Leu Trp Met Ile Gly His Glu Cys Gly HisHis Ser Phe 90 95 100 agt aat tac cgt tgg gtc gac gat aca gtc ggt tttcta atc cat acg 392 Ser Asn Tyr Arg Trp Val Asp Asp Thr Val Gly Phe LeuIle His Thr 105 110 115 gcc acc ctc act ccc tat ttt tcc ttc aaa tat agccac cgt aat cac 440 Ala Thr Leu Thr Pro Tyr Phe Ser Phe Lys Tyr Ser HisArg Asn His 120 125 130 cat gca cac acc aat tcc atg gaa tac gac gag gttcat atc ccg aaa 488 His Ala His Thr Asn Ser Met Glu Tyr Asp Glu Val HisIle Pro Lys 135 140 145 150 cgc aaa tca gaa gct ctc tac ttt gaa ttt ctgggc aac aac cca atc 536 Arg Lys Ser Glu Ala Leu Tyr Phe Glu Phe Leu GlyAsn Asn Pro Ile 155 160 165 ggc tta atg atc acc atg cta tgt aaa ctg actttc gga tat gca gct 584 Gly Leu Met Ile Thr Met Leu Cys Lys Leu Thr PheGly Tyr Ala Ala 170 175 180 tac att atg ttc aat tac aca ggt aag aag cacaaa tct ggg ggc tta 632 Tyr Ile Met Phe Asn Tyr Thr Gly Lys Lys His LysSer Gly Gly Leu 185 190 195 gcg agc cac ttc tac cca caa agc cct ctc tttaac gac agc gaa cgt 680 Ala Ser His Phe Tyr Pro Gln Ser Pro Leu Phe AsnAsp Ser Glu Arg 200 205 210 aac cat gtt ttg ttc tct gac atc ggg att tgcatc gtc ttg tac gcg 728 Asn His Val Leu Phe Ser Asp Ile Gly Ile Cys IleVal Leu Tyr Ala 215 220 225 230 tgt tac cgt att gtg acg gtc aca ggg gcaatg ccg gca ttt tat gtg 776 Cys Tyr Arg Ile Val Thr Val Thr Gly Ala MetPro Ala Phe Tyr Val 235 240 245 tac ggt att cct tgg gtt ata atg agt gctatt ctc ttt gca gca act 824 Tyr Gly Ile Pro Trp Val Ile Met Ser Ala IleLeu Phe Ala Ala Thr 250 255 260 tat tta caa cac act cat cct tca atc cctcat tat gat aca acg gag 872 Tyr Leu Gln His Thr His Pro Ser Ile Pro HisTyr Asp Thr Thr Glu 265 270 275 tgg aac tgg ctt aga ggg gct tta tcg acaatt gat aga gat tta ggg 920 Trp Asn Trp Leu Arg Gly Ala Leu Ser Thr IleAsp Arg Asp Leu Gly 280 285 290 ttc ttc aac atg aac aaa aca cat tat catgtt atc cac cat ttg ttt 968 Phe Phe Asn Met Asn Lys Thr His Tyr His ValIle His His Leu Phe 295 300 305 310 cct gtc att ccg gaa tac cat gca caagag gca acc gag gcc atc aag 1016 Pro Val Ile Pro Glu Tyr His Ala Gln GluAla Thr Glu Ala Ile Lys 315 320 325 ccc atc tta ggt caa tat tac aag tatgat ggt act ccg ttt cta aag 1064 Pro Ile Leu Gly Gln Tyr Tyr Lys Tyr AspGly Thr Pro Phe Leu Lys 330 335 340 gcc ttg tgg aga gaa atg aag gag tgtatt tat gta gaa tcc gat gaa 1112 Ala Leu Trp Arg Glu Met Lys Glu Cys IleTyr Val Glu Ser Asp Glu 345 350 355 ggt cag aag aaa caa ggt att tat tggttt aaa aat aag act 1154 Gly Gln Lys Lys Gln Gly Ile Tyr Trp Phe Lys AsnLys Thr 360 365 370 tgaagtttaa cataatctgg actacgttta attttgtgccatcagtacgt acggtgttag 1214 ttttgttgtg ttttcatttt tcgtattttg tgtgatggtgtaatgtaata taattcagta 1274 taataaagga gttatccttt gatgggttta aaaaaaa 13114 372 PRT Calendula officinalis 4 Met Gly Lys Ala Ala Ser Ala Lys LysVal Leu Glu Arg Val Pro Ile 1 5 10 15 Ser Lys Pro Pro Phe Glu Tyr AsnAsp Leu Lys Lys Ala Val Pro Pro 20 25 30 His Cys Phe Ser Arg Pro Leu SerArg Ser Leu Tyr Phe Leu Phe His 35 40 45 Asp Ile Ile Val Thr Cys Ile LeuPhe Tyr Val Ala Ser Asn Tyr Ile 50 55 60 His Met Leu Pro Arg Phe Leu SerCys Ile Val Trp Pro Val Tyr Trp 65 70 75 80 Ile Ser Gln Gly Val Phe LeuGly Arg Leu Trp Met Ile Gly His Glu 85 90 95 Cys Gly His His Ser Phe SerAsn Tyr Arg Trp Val Asp Asp Thr Val 100 105 110 Gly Phe Leu Ile His ThrAla Thr Leu Thr Pro Tyr Phe Ser Phe Lys 115 120 125 Tyr Ser His Arg AsnHis His Ala His Thr Asn Ser Met Glu Tyr Asp 130 135 140 Glu Val His IlePro Lys Arg Lys Ser Glu Ala Leu Tyr Phe Glu Phe 145 150 155 160 Leu GlyAsn Asn Pro Ile Gly Leu Met Ile Thr Met Leu Cys Lys Leu 165 170 175 ThrPhe Gly Tyr Ala Ala Tyr Ile Met Phe Asn Tyr Thr Gly Lys Lys 180 185 190His Lys Ser Gly Gly Leu Ala Ser His Phe Tyr Pro Gln Ser Pro Leu 195 200205 Phe Asn Asp Ser Glu Arg Asn His Val Leu Phe Ser Asp Ile Gly Ile 210215 220 Cys Ile Val Leu Tyr Ala Cys Tyr Arg Ile Val Thr Val Thr Gly Ala225 230 235 240 Met Pro Ala Phe Tyr Val Tyr Gly Ile Pro Trp Val Ile MetSer Ala 245 250 255 Ile Leu Phe Ala Ala Thr Tyr Leu Gln His Thr His ProSer Ile Pro 260 265 270 His Tyr Asp Thr Thr Glu Trp Asn Trp Leu Arg GlyAla Leu Ser Thr 275 280 285 Ile Asp Arg Asp Leu Gly Phe Phe Asn Met AsnLys Thr His Tyr His 290 295 300 Val Ile His His Leu Phe Pro Val Ile ProGlu Tyr His Ala Gln Glu 305 310 315 320 Ala Thr Glu Ala Ile Lys Pro IleLeu Gly Gln Tyr Tyr Lys Tyr Asp 325 330 335 Gly Thr Pro Phe Leu Lys AlaLeu Trp Arg Glu Met Lys Glu Cys Ile 340 345 350 Tyr Val Glu Ser Asp GluGly Gln Lys Lys Gln Gly Ile Tyr Trp Phe 355 360 365 Lys Asn Lys Thr 3705 387 PRT Glycine max 5 Met Gly Leu Ala Lys Glu Thr Thr Met Gly Gly ArgGly Arg Val Ala 1 5 10 15 Lys Val Glu Val Gln Gly Lys Lys Pro Leu SerArg Val Pro Asn Thr 20 25 30 Lys Pro Pro Phe Thr Val Gly Gln Leu Lys LysAla Ile Pro Pro His 35 40 45 Cys Phe Gln Arg Ser Leu Leu Thr Ser Phe SerTyr Val Val Tyr Asp 50 55 60 Leu Ser Phe Ala Phe Ile Phe Tyr Ile Ala ThrThr Tyr Phe His Leu 65 70 75 80 Leu Pro Gln Pro Phe Ser Leu Ile Ala TrpPro Ile Tyr Trp Val Leu 85 90 95 Gln Gly Cys Leu Leu Thr Gly Val Trp ValIle Ala His Glu Cys Gly 100 105 110 His His Ala Phe Ser Lys Tyr Gln TrpVal Asp Asp Val Val Gly Leu 115 120 125 Thr Leu His Ser Thr Leu Leu ValPro Tyr Phe Ser Trp Lys Ile Ser 130 135 140 His Arg Arg His His Ser AsnThr Gly Ser Leu Asp Arg Asp Glu Val 145 150 155 160 Phe Val Pro Lys ProLys Ser Lys Val Ala Trp Phe Ser Lys Tyr Leu 165 170 175 Asn Asn Pro LeuGly Arg Ala Val Ser Leu Leu Val Thr Leu Thr Ile 180 185 190 Gly Trp ProMet Tyr Leu Ala Phe Asn Val Ser Gly Arg Pro Tyr Asp 195 200 205 Ser PheAla Ser His Tyr His Pro Tyr Ala Pro Ile Tyr Ser Asn Arg 210 215 220 GluArg Leu Leu Ile Tyr Val Ser Asp Val Ala Leu Phe Ser Val Thr 225 230 235240 Tyr Ser Leu Tyr Arg Val Ala Thr Leu Lys Gly Leu Val Trp Leu Leu 245250 255 Cys Val Tyr Gly Val Pro Leu Leu Ile Val Asn Gly Phe Leu Val Thr260 265 270 Ile Thr Tyr Leu Gln His Thr His Phe Ala Leu Pro His Tyr AspSer 275 280 285 Ser Glu Trp Asp Trp Leu Lys Gly Ala Leu Ala Thr Met AspArg Asp 290 295 300 Tyr Gly Ile Leu Asn Lys Val Phe His His Ile Thr AspThr His Val 305 310 315 320 Ala His His Leu Phe Ser Thr Met Pro His TyrHis Ala Met Glu Ala 325 330 335 Thr Asn Ala Ile Lys Pro Ile Leu Gly GluTyr Tyr Gln Phe Asp Asp 340 345 350 Thr Pro Phe Tyr Lys Ala Leu Trp ArgGlu Ala Arg Glu Cys Leu Tyr 355 360 365 Val Glu Pro Asp Glu Gly Thr SerGlu Lys Gly Val Tyr Trp Tyr Arg 370 375 380 Asn Lys Tyr 385 6 387 PRTRicinus communis 6 Met Gly Gly Gly Gly Arg Met Ser Thr Val Ile Thr SerAsn Asn Ser 1 5 10 15 Glu Lys Lys Gly Gly Ser Ser His Leu Lys Arg AlaPro His Thr Lys 20 25 30 Pro Pro Phe Thr Leu Gly Asp Leu Lys Arg Ala IlePro Pro His Cys 35 40 45 Phe Glu Arg Ser Phe Val Arg Ser Phe Ser Tyr ValAla Tyr Asp Val 50 55 60 Cys Leu Ser Phe Leu Phe Tyr Ser Ile Ala Thr AsnPhe Phe Pro Tyr 65 70 75 80 Ile Ser Ser Pro Leu Ser Tyr Val Ala Trp LeuVal Tyr Trp Leu Phe 85 90 95 Gln Gly Cys Ile Leu Thr Gly Leu Trp Val IleGly His Glu Cys Gly 100 105 110 His His Ala Phe Ser Glu Tyr Gln Leu AlaAsp Asp Ile Val Gly Leu 115 120 125 Ile Val His Ser Ala Leu Leu Val ProTyr Phe Ser Trp Lys Tyr Ser 130 135 140 His Arg Arg His His Ser Asn IleGly Ser Leu Glu Arg Asp Glu Val 145 150 155 160 Phe Val Pro Lys Ser LysSer Lys Ile Ser Trp Tyr Ser Lys Tyr Ser 165 170 175 Asn Asn Pro Pro GlyArg Val Leu Thr Leu Ala Ala Thr Leu Leu Leu 180 185 190 Gly Trp Pro LeuTyr Leu Ala Phe Asn Val Ser Gly Arg Pro Tyr Asp 195 200 205 Arg Phe AlaCys His Tyr Asp Pro Tyr Gly Pro Ile Phe Ser Glu Arg 210 215 220 Glu ArgLeu Gln Ile Tyr Ile Ala Asp Leu Gly Ile Phe Ala Thr Thr 225 230 235 240Phe Val Leu Tyr Gln Ala Thr Met ala Lys Gly Leu Ala Trp Val Met 245 250255 Arg Ile Tyr Gly Val Pro Leu Leu Ile Val Asn Cys Phe Leu Val Met 260265 270 Ile Thr Tyr Leu Gln His Thr His Pro Ala Ile Pro Arg Tyr Gly Ser275 280 285 Ser Glu Trp Asp Trp Leu Arg Gly Ala Met Val Thr Val Asp ArgAsp 290 295 300 Tyr Gly Val Leu Asn Lys Val Phe His Asn Ile Ala Asp ThrHis Val 305 310 315 320 Ala His His Leu Phe Ala Thr Val Pro His Tyr HisAla Met Glu Ala 325 330 335 Thr Lys Ala Ile Lys Pro Ile Met Gly Glu TyrTyr Arg Tyr Asp Gly 340 345 350 Thr Pro Phe Tyr Lys Ala Leu Trp Arg GluAla Lys Glu Cys Leu Phe 355 360 365 Val Glu Pro Asp Glu Gly Ala Pro ThrGln Gly Val Phe Trp Tyr Arg 370 375 380 Asn Lys Tyr 385 7 383 PRTImpatiens balsamina 7 Met Gly Glu Val Gly Pro Thr Asn Arg Thr Lys ThrLys Leu Asp Lys 1 5 10 15 Gln Gln Glu Ser Glu Asn Arg Val Pro His GluPro Pro Pro Phe Thr 20 25 30 Leu Ser Asp Leu Lys Lys Ala Ile Pro Pro HisCys Phe Glu Arg Ser 35 40 45 Leu Val Lys Ser Phe Tyr His Val Ile His AspIle Ile Ile Leu Ser 50 55 60 Phe Phe Tyr Tyr Val Ala Ala Asn Tyr Ile ProMet Leu Pro Gln Asn 65 70 75 80 Leu Arg Tyr Val Ala Trp Pro Ile Tyr TrpAla Ile Gln Gly Cys Val 85 90 95 Gln Leu Gly Ile Leu Val Leu Gly His GluCys Gly His His Ala Phe 100 105 110 Ser Asp Tyr Gln Trp Val Asp Asp MetVal Gly Phe Val Leu His Ser 115 120 125 Ser Gln Leu Ile Pro Tyr Phe SerTrp Lys His Ser His Arg Arg His 130 135 140 His Ser Asn Thr Ala Ser IleGlu Arg Asp Glu Val Tyr Pro Pro Ala 145 150 155 160 Tyr Lys Asn Asp LeuPro Trp Phe Ala Lys Tyr Leu Arg Asn Pro Val 165 170 175 Gly Arg Phe LeuMet Ile Phe Gly Ala Leu Leu Phe Gly Trp Pro Ser 180 185 190 Tyr Leu LeuPhe Asn Ala Asn Gly Arg Leu Tyr Asp Arg Phe Ala Ser 195 200 205 His TyrAsp Pro Gln Ser Pro Ile Phe Asn Asn Arg Glu Arg Leu Gln 210 215 220 ValIle Ala Ser Asp Val Gly Leu Val Phe Ala Tyr Phe Val Leu Tyr 225 230 235240 Lys Ile Ala Leu Ala Lys Gly Phe Val Trp Leu Ile Cys Val Tyr Gly 245250 255 Val Pro Tyr Val Ile Leu Asn Gly Leu Ile Val Leu Ile Thr Phe Leu260 265 270 Gln His Thr His Pro Asn Leu Pro Arg Tyr Asp Leu Ser Glu TrpAsp 275 280 285 Trp Leu Arg Gly Ala Leu Ser Thr Val Asp Arg Asp Tyr GlyMet Leu 290 295 300 Asn Lys Val Phe His Asn Val Thr Asp Thr His Leu ValHis His Leu 305 310 315 320 Phe Thr Thr Met Pro His Tyr Arg Ala Lys GluAla Thr Glu Val Ile 325 330 335 Lys Pro Ile Leu Gly Asp Tyr Tyr Lys PheAsp Asp Thr Pro Phe Leu 340 345 350 Lys Ala Leu Trp Lys Asp Met Gly LysCys Ile Tyr Val Glu Ser Asp 355 360 365 Val Pro Gly Lys Asn Lys Gly ValTyr Trp Tyr Asn Asn Asp Ile 370 375 380 8 399 PRT Momordica charantia 8Met Gly Gly Arg Gly Ala Ile Gly Val Leu Arg Asn Gly Gly Gly Pro 1 5 1015 Lys Lys Lys Met Gly Pro Gly Gln Gly Leu Gly Pro Gly Glu Arg Ile 20 2530 Thr His Ala Arg Pro Pro Phe Ser Ile Ser Gln Ile Lys Lys Ala Ile 35 4045 Pro Pro His Cys Phe Gln Arg Ser Leu Arg Arg Ser Phe Ser Tyr Leu 50 5560 Leu Ser Asp Ile Ala Leu Val Ser Ala Phe Tyr Tyr Val Ala Asp Thr 65 7075 80 Tyr Phe His Arg Leu Pro His Pro Leu Leu His Tyr Leu Ala Trp Pro 8590 95 Val Tyr Trp Phe Cys Gln Gly Ala Val Leu Thr Gly Met Trp Gly Ile100 105 110 Ala His Asp Cys Gly His His Ala Phe Ser Asp Tyr Gln Leu ValAsp 115 120 125 Asp Val Val Gly Phe Leu Ile His Ser Leu Val Phe Val ProTyr Phe 130 135 140 Ser Phe Lys Ile Ser His Arg Arg His His Ser Asn ThrSer Ser Val 145 150 155 160 Asp Arg Asp Glu Val Phe Val Pro Lys Pro LysAla Lys Met Pro Trp 165 170 175 Tyr Phe Lys Tyr Leu Thr Asn Pro Pro AlaArg Val Phe Ile Ile Phe 180 185 190 Ile Thr Leu Thr Leu Gly Trp Pro MetTyr Leu Thr Phe Asn Ile Ser 195 200 205 Gly Arg Tyr Tyr Gly Arg Phe ThrSer His Phe Asp Pro Asn Ser Pro 210 215 220 Ile Phe Ser Pro Lys Glu ArgVal Leu Val His Ile Ser Asn Ala Gly 225 230 235 240 Leu Val Ala Thr GlyTyr Leu Leu Tyr Arg Ile Ala Met ala Lys Gly 245 250 255 Val Gly Trp LeuIle Arg Leu Tyr Gly Val Pro Leu Ile Val Leu Asn 260 265 270 Ala Cys ValVal Leu Ile Thr Ala Leu Gln His Thr His Pro Ser Phe 275 280 285 Pro TyrTyr Asp Ser Thr Glu Trp Asp Trp Leu Arg Gly Asn Leu Val 290 295 300 ThrVal Asp Arg Asp Tyr Gly Pro Ile Met Asn Arg Val Phe His His 305 310 315320 Ile Thr Asp Thr His Val Val His His Leu Phe Pro Ser Met Pro His 325330 335 Tyr Asn Gly Lys Glu Ala Thr Val Ala Ala Lys Arg Ile Leu Gly Glu340 345 350 Tyr Tyr Gln Phe Asp Gly Thr Pro Ile Trp Lys Ala Ala Trp ArgGlu 355 360 365 Phe Arg Glu Cys Val Tyr Val Glu Pro Asp Glu Asp Asp GlyAla Thr 370 375 380 Ser Gly Ser Ser Ser Lys Gly Val Phe Trp Tyr His AsnLys Leu 385 390 395 9 387 PRT Chrysobalanus icaco 9 Met Gly Ala Gly GlyGln Lys Thr Phe Pro Arg Leu Glu Glu Glu Glu 1 5 10 15 Lys Gln Gln GlnAla Ala Ala Ala Gly Phe Lys Arg Ile Pro Thr Thr 20 25 30 Lys Pro Pro PheThr Leu Ser Asp Leu Lys Lys Ala Ile Pro Pro His 35 40 45 Cys Phe Gln ArgSer Leu Leu Arg Ser Phe Ser Tyr Val Phe Ile Asp 50 55 60 Leu Thr Ile IleSer Ile Leu Gly Tyr Ile Gly Ala Thr Tyr Ile Cys 65 70 75 80 Leu Leu ProPro Pro Ser Lys Tyr Leu Ala Trp Leu Leu Tyr Trp Ala 85 90 95 Val Gln GlyCys Phe Phe Thr Gly Ala Trp Ala Leu Ala His Asp Cys 100 105 110 Gly HisHis Ala Phe Ser Asp Tyr Gln Trp Ile Asp Asp Ala Val Gly 115 120 125 MetVal Leu His Ser Thr Leu Met Val Pro Tyr Phe Ser Phe Lys Tyr 130 135 140Ser His Arg Arg His His Ser Asn Ile Asn Ser Leu Glu Arg Asp Glu 145 150155 160 Val Phe Val Pro Arg Pro Lys Ser Lys Ile Lys Trp Tyr Cys Ser Lys165 170 175 Tyr Leu Asn Asn Pro Leu Gly Arg Val Leu Thr Leu Ala Val ThrLeu 180 185 190 Ile Leu Gly Trp Pro Met Tyr Leu Ala Leu Asn Ala Ser GlyArg Asp 195 200 205 Tyr Asp Arg Phe Val Ser His Phe Tyr Pro Tyr Gly ProIle Tyr Asn 210 215 220 Asp Arg Glu Arg Leu Gln Ile Tyr Ile Ser Asp AlaGly Ile Phe Ile 225 230 235 240 Val Ser Tyr Val Leu Tyr Gln Val Ala LeuAla Lys Gly Leu Pro Trp 245 250 255 Leu Ile Cys Ile Tyr Gly Val Pro LeuPhe Val Asn Asn Ala Leu Val 260 265 270 Val Thr Ile Thr Tyr Leu Gln HisThr His Pro Glu Leu Pro Arg Tyr 275 280 285 Gly Asn Ser Glu Trp Asp TrpPhe Lys Gly Thr Leu Ala Thr Val Asp 290 295 300 Arg Asp Met Gly Pro LeuLeu Asn Trp Ala Thr His His Val Ser Asp 305 310 315 320 Thr His Tyr ValHis His Leu Phe Ser Thr Met Pro His Tyr His Gly 325 330 335 Val Glu AlaThr Lys Ala Val Lys Pro Met Leu Gly Glu Tyr Tyr Arg 340 345 350 Phe AspPro Thr Pro Leu Tyr Lys Ala Leu Trp Arg Glu Ala Lys Glu 355 360 365 CysLeu Phe Val Glu Pro Asp Ser Lys Ser Pro Gly Val Phe Trp Phe 370 375 380Asp Lys Phe 385 10 1361 DNA Dimorphotheca sinuata 10 ggcacgagctacaagaaacc ttcaacaaca aaatgggagc aggaggttgc atctctgtct 60 ccgaaaccaaacccaaccaa aaaaacagtc tcgaacgagc cccttacgac aaaccgcctt 120 tcaccatcagcgacctcaaa aaagccatcc ctccccactt atttaaacgt tccttaatcc 180 gttcattatcttacgtcgcc tctgacctca ccgtagcctt cctcctctac cacgccacca 240 cctacttccaccacctcccg caaccgttca ccgccctcgc atggctagct tattgggtag 300 cccaagggtgtgtgctcacc ggagtttggg tcataggcca tgaatgtggt caccatggac 360 ttagcgaatatcgaggggtt gacgacacgg ttggctacat actccactcg tctttactcg 420 tcccgtatttctcgtggaaa tatagtcacc gtcgccacca ctccaacacc ggatcactcg 480 accgcgatgaagtattcgtc ccaaaaccaa gatcaaaaat atcatggtat tcaaagtact 540 ttaacaacccggtcggacga atcggggttc tattcatcac gctcactctc ggctggccgt 600 tatacttaactttcaatgtt tccggaagac cctacgaccg tttcgcgtgc cactattctc 660 ctaacagcccgatatacaac aaccgtgaac gcttccaaat ttatctttcc gatatcggga 720 tcgtcatcacgtcattagtc cttttacgtg ctgcgatggt gaaagggttg gtttggttaa 780 tttgcgtctacggggtcccg ttaatgataa cgaacgggtt tcttgtattg gttacgtatc 840 ttcaacatactcacccttca ttgcctcatt acgataactc ggaatgggag tggttaaagg 900 gagcattagtgactgtggac cgtgattttg gtgtgttaaa cacggtgttt catcacgcta 960 cggatggacacattgtgcat catttgttcc caacaatacc acattataac gcgatggaag 1020 caactaaagcggtgaagcct ttgatggggg agtattatca gtatgacgca actccgtttt 1080 atgtagcgatgtggagagag gcgaaggagt gtttgtttgt cgatcgggat gagggggaga 1140 aaggaggtgtgttttggtac aagaataaaa tgtaatgtgt gtatgtgtga gtttttagtt 1200 taggtagtttatgagtatgg ctggtgtttt tagtaatgtt gcgtgtgtgt gtgtgttcga 1260 accttgtgtatgyggttgtg tyatgtgtat gataaatgta atgtacctca ttaaaaggac 1320 ttatgttatctaaaataaga atgtktcttg ttggttatcg g 1361 11 380 PRT Dimorphotheca sinuata11 Met Gly Ala Gly Gly Cys Ile Ser Val Ser Glu Thr Lys Pro Asn Gln 1 510 15 Lys Asn Ser Leu Glu Arg Ala Pro Tyr Asp Lys Pro Pro Phe Thr Ile 2025 30 Ser Asp Leu Lys Lys Ala Ile Pro Pro His Leu Phe Lys Arg Ser Leu 3540 45 Ile Arg Ser Leu Ser Tyr Val Ala Ser Asp Leu Thr Val Ala Phe Leu 5055 60 Leu Tyr His Ala Thr Thr Tyr Phe His His Leu Pro Gln Pro Phe Thr 6570 75 80 Ala Leu Ala Trp Leu Ala Tyr Trp Val Ala Gln Gly Cys Val Leu Thr85 90 95 Gly Val Trp Val Ile Gly His Glu Cys Gly His His Gly Leu Ser Glu100 105 110 Tyr Arg Gly Val Asp Asp Thr Val Gly Tyr Ile Leu His Ser SerLeu 115 120 125 Leu Val Pro Tyr Phe Ser Trp Lys Tyr Ser His Arg Arg HisHis Ser 130 135 140 Asn Thr Gly Ser Leu Asp Arg Asp Glu Val Phe Val ProLys Pro Arg 145 150 155 160 Ser Lys Ile Ser Trp Tyr Ser Lys Tyr Phe AsnAsn Pro Val Gly Arg 165 170 175 Ile Gly Val Leu Phe Ile Thr Leu Thr LeuGly Trp Pro Leu Tyr Leu 180 185 190 Thr Phe Asn Val Ser Gly Arg Pro TyrAsp Arg Phe Ala Cys His Tyr 195 200 205 Ser Pro Asn Ser Pro Ile Tyr AsnAsn Arg Glu Arg Phe Gln Ile Tyr 210 215 220 Leu Ser Asp Ile Gly Ile ValIle Thr Ser Leu Val Leu Leu Arg Ala 225 230 235 240 Ala Met Val Lys GlyLeu Val Trp Leu Ile Cys Val Tyr Gly Val Pro 245 250 255 Leu Met Ile ThrAsn Gly Phe Leu Val Leu Val Thr Tyr Leu Gln His 260 265 270 Thr His ProSer Leu Pro His Tyr Asp Asn Ser Glu Trp Glu Trp Leu 275 280 285 Lys GlyAla Leu Val Thr Val Asp Arg Asp Phe Gly Val Leu Asn Thr 290 295 300 ValPhe His His Ala Thr Asp Gly His Ile Val His His Leu Phe Pro 305 310 315320 Thr Ile Pro His Tyr Asn Ala Met Glu Ala Thr Lys Ala Val Lys Pro 325330 335 Leu Met Gly Glu Tyr Tyr Gln Tyr Asp Ala Thr Pro Phe Tyr Val Ala340 345 350 Met Trp Arg Glu Ala Lys Glu Cys Leu Phe Val Asp Arg Asp GluGly 355 360 365 Glu Lys Gly Gly Val Phe Trp Tyr Lys Asn Lys Met 370 375380 12 1337 DNA Dimorphotheca sinuata 12 gggggatggg agcatctgaggagatgaagg tcttggaacg agttccagtc tcaaaacctc 60 cattcgagta caatgatctgaagaaagcag taccaccaca ttgttttaca cgatcacttt 120 cactctcgtt ttattacctgttttatgacc taataaaagt atgtatcctt ttctacgtag 180 cctcaaaata cattcctatgcttccttata gcctttcctg cattgtatgg cctctttact 240 ggttcttcca aggagcttttctaggcagat tgtggatgat tggccatgaa tgcgggcatc 300 atagctttag taattatcgttggttagacg ataccgttgg gttcttggtc cacactgcca 360 ccctcactcc atatttttctttcaaataca gtcaccgtaa tcaccatgca cacaccaatt 420 ccttggagta tgacgaggttcatgtcccta agattaggaa atttaaatcc gaacatctct 480 actctgaatt tctcaccaacaacccatttg gcttagtggt caacatggta tttgaactca 540 cttttggata cccatcttacttaatattca attattcagg tagaaagctt actcaagctg 600 gttttgcaag tcacttgtacccacaaagcc caatcttcaa cgatagtgaa cgtaatcatg 660 tgtttttctc tgatgttggtatttgcattg tgttatacgc attataccgc atagcgatag 720 ccaaaggcgc aatgctagtgttgtatgtgt atggtcttcc ttgggttgta atgagtgctt 780 tcatcttttc ccttacttatttacaacaca ctcatccttc catccctcac tatgattcaa 840 ctgagtggaa ttggctcagaggtgctttat cctcaatcga cagagaatta gcaggggcct 900 tcaacatcaa aaaaacacattatcatgttg tgcaccattt gtttcccttt attccagaat 960 accatgcaca cgacgccaccgaggccctta agcccatctt aggcccatat tacaagtatg 1020 atggcactcc gttttataaggcgttgtgga gagaaatgaa ggactgtctt tatgttgaat 1080 ctgatgatgg ccccaacaaaactggtgttt actggttcaa aactaagact tgattaatca 1140 gctggcgtgt caccagcccgcccgggttcg ggttagggtt agggttaatt tcattgcagt 1200 aattttcttt ttcatttctttttatttttc ttttatattg ttctcagtac ctgtatgttt 1260 gggttattgt gtaatgtataataattcagt ttaataaaac cctttatatt ttgatattaa 1320 aaaaaaaaaa aaaaaaa 133713 375 PRT Dimorphotheca sinuata 13 Met Gly Ala Ser Glu Glu Met Lys ValLeu Glu Arg Val Pro Val Ser 1 5 10 15 Lys Pro Pro Phe Glu Tyr Asn AspLeu Lys Lys Ala Val Pro Pro His 20 25 30 Cys Phe Thr Arg Ser Leu Ser LeuSer Phe Tyr Tyr Leu Phe Tyr Asp 35 40 45 Leu Ile Lys Val Cys Ile Leu PheTyr Val Ala Ser Lys Tyr Ile Pro 50 55 60 Met Leu Pro Tyr Ser Leu Ser CysIle Val Trp Pro Leu Tyr Trp Phe 65 70 75 80 Phe Gln Gly Ala Phe Leu GlyArg Leu Trp Met Ile Gly His Glu Cys 85 90 95 Gly His His Ser Phe Ser AsnTyr Arg Trp Leu Asp Asp Thr Val Gly 100 105 110 Phe Leu Val His Thr AlaThr Leu Thr Pro Tyr Phe Ser Phe Lys Tyr 115 120 125 Ser His Arg Asn HisHis Ala His Thr Asn Ser Leu Glu Tyr Asp Glu 130 135 140 Val His Val ProLys Ile Arg Lys Phe Lys Ser Glu His Leu Tyr Ser 145 150 155 160 Glu PheLeu Thr Asn Asn Pro Phe Gly Leu Val Val Asn Met Val Phe 165 170 175 GluLeu Thr Phe Gly Tyr Pro Ser Tyr Leu Ile Phe Asn Tyr Ser Gly 180 185 190Arg Lys Leu Thr Gln Ala Gly Phe Ala Ser His Leu Tyr Pro Gln Ser 195 200205 Pro Ile Phe Asn Asp Ser Glu Arg Asn His Val Phe Phe Ser Asp Val 210215 220 Gly Ile Cys Ile Val Leu Tyr Ala Leu Tyr Arg Ile Ala Ile Ala Lys225 230 235 240 Gly Ala Met Leu Val Leu Tyr Val Tyr Gly Leu Pro Trp ValVal Met 245 250 255 Ser Ala Phe Ile Phe Ser Leu Thr Tyr Leu Gln His ThrHis Pro Ser 260 265 270 Ile Pro His Tyr Asp Ser Thr Glu Trp Asn Trp LeuArg Gly Ala Leu 275 280 285 Ser Ser Ile Asp Arg Glu Leu Ala Gly Ala PheAsn Ile Lys Lys Thr 290 295 300 His Tyr His Val Val His His Leu Phe ProPhe Ile Pro Glu Tyr His 305 310 315 320 Ala His Asp Ala Thr Glu Ala LeuLys Pro Ile Leu Gly Pro Tyr Tyr 325 330 335 Lys Tyr Asp Gly Thr Pro PheTyr Lys Ala Leu Trp Arg Glu Met Lys 340 345 350 Asp Cys Leu Tyr Val GluSer Asp Asp Gly Pro Asn Lys Thr Gly Val 355 360 365 Tyr Trp Phe Lys ThrLys Thr 370 375 14 378 PRT Helianthus annuus 14 Met Gly Ala Gly Glu TyrThr Ser Val Thr Asn Glu Asn Asn Pro Leu 1 5 10 15 Asp Arg Val Pro HisAla Lys Pro Pro Phe Thr Ile Gly Asp Leu Lys 20 25 30 Lys Ala Ile Pro ProHis Cys Phe Gln Arg Ser Leu Thr Arg Ser Phe 35 40 45 Ser Tyr Val Leu SerAsp Leu Thr Ile Thr Ala Val Leu Tyr His Ile 50 55 60 Ala Thr Thr Tyr PheHis His Leu Pro Thr Pro Leu Ser Ser Ile Ala 65 70 75 80 Trp Ala Ser TyrTrp Val Val Gln Gly Cys Val Leu Thr Gly Val Trp 85 90 95 Val Ile Ala HisGlu Cys Gly His His Ala Phe Ser Asp Tyr Gln Trp 100 105 110 Val Asp AspThr Val Gly Phe Val Leu His Ser Ser Leu Leu Val Pro 115 120 125 Tyr PheSer Trp Lys Tyr Ser His His Arg His His Ser Asn Thr Gly 130 135 140 SerLeu Glu Arg Asp Glu Val Phe Val Pro Lys Ser Arg Ser Lys Val 145 150 155160 Pro Trp Tyr Ser Lys Tyr Phe Asn Asn Thr Val Gly Arg Ile Val Ser 165170 175 Met Phe Val Thr Leu Thr Leu Gly Trp Pro Leu Tyr Leu Ala Phe Asn180 185 190 Val Ser Gly Arg Pro Tyr Asp Arg Phe Ala Cys His Tyr Val ProThr 195 200 205 Ser Pro Met Tyr Asn Glu Arg Lys Arg Tyr Gln Ile Val MetSer Asp 210 215 220 Ile Gly Ile Val Ile Thr Ser Phe Ile Leu Tyr Arg ValAla Met ala 225 230 235 240 Lys Gly Leu Val Trp Val Ile Cys Val Tyr GlyVal Pro Leu Met Val 245 250 255 Val Asn Ala Phe Leu Val Leu Ile Thr TyrLeu Gln His Thr His Pro 260 265 270 Gly Leu Pro His Tyr Asp Ser Ser GluTrp Glu Trp Leu Lys Gly Ala 275 280 285 Leu Ala Thr Val Asp Arg Asp TyrGly Val Leu Asn Lys Val Phe His 290 295 300 His Ile Thr Asp Thr His ValVal His His Leu Phe Ser Thr Met Pro 305 310 315 320 His Tyr Asn Ala MetGlu Ala Gln Lys Ala Leu Arg Pro Val Leu Gly 325 330 335 Glu Tyr Tyr ArgPhe Asp Lys Thr Pro Phe Tyr Val Ala Met Trp Arg 340 345 350 Glu Met LysGlu Cys Leu Phe Val Glu Gln Asp Asp Glu Gly Lys Gly 355 360 365 Gly ValPhe Trp Tyr Lys Asn Lys Met Asn 370 375 15 383 PRT Borago officinalis 15Met Gly Gly Gly Gly Arg Met Pro Val Pro Thr Lys Gly Lys Lys Ser 1 5 1015 Lys Ser Asp Val Phe Gln Arg Val Pro Ser Glu Lys Pro Pro Phe Thr 20 2530 Val Gly Asp Leu Lys Lys Val Ile Pro Pro His Cys Phe Gln Arg Ser 35 4045 Val Leu His Ser Phe Ser Tyr Val Val Tyr Asp Leu Val Ile Ala Ala 50 5560 Leu Phe Phe Tyr Thr Ala Ser Arg Tyr Ile His Leu Gln Pro His Pro 65 7075 80 Leu Ser Tyr Val Ala Trp Pro Leu Tyr Trp Phe Cys Gln Gly Ser Val 8590 95 Leu Thr Gly Val Trp Val Ile Ala His Glu Cys Gly His His Ala Phe100 105 110 Ser Asp Tyr Gln Trp Leu Asp Asp Thr Val Gly Leu Leu Leu HisSer 115 120 125 Ala Leu Leu Val Pro Tyr Phe Ser Trp Lys Tyr Ser His ArgArg His 130 135 140 His Ser Asn Thr Gly Ser Leu Glu Arg Asp Glu Val PheVal Pro Lys 145 150 155 160 Lys Arg Ser Gly Ile Ser Trp Ser Ser Glu TyrLeu Asn Asn Pro Pro 165 170 175 Gly Arg Val Leu Val Leu Leu Val Gln LeuThr Leu Gly Trp Pro Leu 180 185 190 Tyr Leu Met Phe Asn Val Ser Gly ArgPro Tyr Asp Arg Phe Ala Cys 195 200 205 His Phe Asp Pro Lys Ser Pro IleTyr Asn Asp Arg Glu Arg Leu Gln 210 215 220 Ile Tyr Ile Ser Asp Ala GlyIle Val Ala Val Met Tyr Gly Leu Tyr 225 230 235 240 Arg Leu Val Ala AlaLys Gly Val Ala Trp Val Val Cys Tyr Tyr Gly 245 250 255 Val Pro Leu LeuVal Val Asn Gly Phe Leu Val Leu Ile Thr Tyr Leu 260 265 270 Gln His ThrGln Pro Ser Leu Pro His Tyr Asp Ser Ser Glu Trp Asp 275 280 285 Trp LeuLys Gly Ala Leu Ala Thr Val Asp Arg Asp Tyr Gly Phe Leu 290 295 300 AsnLys Val Leu His Asn Ile Thr Asp Thr His Val Ala His His Leu 305 310 315320 Phe Ser Thr Met Pro His Tyr His Ala Met Glu Ala Thr Lys Ala Ile 325330 335 Lys Pro Ile Leu Gly Asp Tyr Tyr Gln Cys Asp Arg Thr Pro Val Phe340 345 350 Lys Ala Met Tyr Arg Glu Val Lys Glu Cys Ile Tyr Val Glu AlaAsp 355 360 365 Glu Gly Asp Asn Lys Lys Gly Val Phe Trp Tyr Lys Asn LysLeu 370 375 380 16 30 DNA Artificial Sequence Definition of ArtificialSequence Calendula officinalis PCR primer 16 tttgagctct acacctagctacgtaccatg 30 17 30 DNA Artificial Sequence Definition of ArtificialSequence Calendula officinalis PCR primer 17 tttggatcct cacggtactgatgatggcac 30 18 31 DNA Artificial Sequence Definition of ArtificialSequence Calendula Fad2-1 PCR primer 18 ttgcggccgc tacacctagc tacgtaccatg 31 19 30 DNA Artificial Sequence Definition of Artificial SequenceCalendula Fad2-1 PCR primer 19 ttgcggccgt cacggtactg atgatggcac 30 20 24DNA Artificial Sequence Definition of Artificial Sequence CalendulaFad2-2 PCR primer 20 agcggccgct ataccatggg caag 24 21 22 DNA ArtificialSequence Definition of Artificial Sequence Calendula Fad2-2 PCR primer21 tgcggccgct atgttaaact tc 22 22 30 DNA Artificial Sequence Definitionof Artificial Sequence Calendula Fad2-2 PCR primer 22 tatgcggccgcaaatgggag caggaggttg 30 23 32 DNA Artificial Sequence Definition ofArtificial Sequence Calendula Fad2-2 PCR primer 23 tttgcggccg cattacatcttattcttgta cc 32 24 37 DNA Artificial Sequence Definition of ArtificialSequence Calendula Fad2-2 PCR primer 24 tgcggccgca atgggtggag ggatgggagcatctgag 37 25 28 DNA Artificial Sequence Definition of ArtificialSequence Calendula Fad2-2 PCR primer 25 tagcggccgc tgattaatca agtcttag28

What is claimed is:
 1. A chimeric gene comprising an isolated nucleicacid fragment encoding a plant fatty acid modifying enzyme associatedwith conjugated double bond formation comprising a delta-9 position offatty acids having an amino acid identity of at least 72.5% based on theClustal method of alignment when compared to a polypeptide of SEQ IDNO:2 or 4 wherein said fragment or a functionally equivalent subfragmentthereof or a complement thereof is operably linked to suitableregulatory sequences.
 2. The chimeric gene of claim 1 wherein thenucleic acid fragment is isolated from Calendula officinalis.
 3. Thechimeric gene of claim 1 wherein the plant fatty acid modifying enzymeis associated with the formation of calendic acid.
 4. A transformed hostcell or plant comprising in its genome the chimeric gene of claim
 1. 5.A transformed host cell or plant comprising in its genome the chimericgene of claim
 2. 6. A transformed host cell or plant comprising in itsgenome the chimeric gene of claim
 3. 7. A method of altering the levelof fatty acids in a host cell or plant wherein said fatty acids comprisea modification at a delta-9 position, said method comprising: (a)transforming a host cell or plant with the chimeric gene of claim 1; (b)growing the transformed host cell or plant under conditions suitable forthe expression of the chimeric gene; and (c) selecting those transformedhost cells or plants having altered levels of fatty acids comprising amodified delta-9 position.
 8. A method of altering the level of fattyacids in a host cell or plant wherein said fatty acids comprise amodification at a delta-9 position, said method comprising: (a)transforming a host cell or plant with the chimeric gene of claim 2; (b)growing the transformed host cell or plant under conditions suitable forthe expression of the chimeric gene; and (c) selecting those transformedhost cells or plants having altered levels of fatty acids comprising amodified delta-9 position.
 9. A method of altering the level of fattyacids in a host cell or plant wherein said fatty acids comprise amodification at a delta-9 position, said method comprising: (a)transforming a host cell or plant with the chimeric gene of claim 3; (b)growing the transformed host cell or plant under conditions suitable forthe expression of the chimeric gene; and (c) selecting those transformedhost cells or plants having altered levels of fatty acids comprising amodified delta-9 position.
 10. The method of claim 7, 8, or 9 whereinthe host cell or plant is selected from the group consisting of plantcells and microorganisms.
 11. The method of claim 7, 8, or 9 and whereinthe level of calendic acid is altered.
 12. A method for producing fattyacid modifying enzymes associated with modification of a delta-9position of fatty acids which comprises: (a) transforming a microbialhost cell with the chimeric gene of claim 1; (b) growing the transformedhost cell under conditions suitable for the expression of the chimericgene; and (c) selecting those transformed host cells containing alteredlevels of protein encoded by the chimeric gene.
 13. A method forproducing fatty acid modifying enzymes associated with modification of adelta-9 position of fatty acids which comprises: (a) transforming amicrobial host cell with the chimeric gene of claim 2; (b) growing thetransformed host cell under conditions suitable for the expression ofthe chimeric gene; and (c) selecting those transformed host cellscontaining altered levels of protein encoded by the chimeric gene.
 14. Amethod for producing fatty acid modifying enzymes associated withmodification of a delta-9 position of fatty acids which comprises: (a)transforming a microbial host cell with the chimeric gene of claim 3;(b) growing the transformed host cell under conditions suitable for theexpression of the chimeric gene; and (c) selecting those transformedhost cells containing altered levels of protein encoded by the chimericgene.
 15. The method of claim 12, 13, or 14 wherein the fatty acidmodifying enzyme is associated with the formation of calendic acid ordimorphecolic acid.